WO2019118727A2

WO2019118727A2 - Rescue of the pathology of lrrk2 on lysosmes with snx25 or snx27

Info

Publication number: WO2019118727A2
Application number: PCT/US2018/065451
Authority: WO
Inventors: Asa Abeliovich
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2017-12-14
Filing date: 2018-12-13
Publication date: 2019-06-20
Also published as: WO2019118727A3

Abstract

The present invention relates to methods of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a nucleic acid encoding SNX25, a nucleic acid encoding SNX27, a SNX25 polypeptide, a SNX27 polypeptide, a SNX25 activator or agonist, a SNX27 activator or agonist, or a combination thereof.

Description

RESCUE OF THE PATHOLOGY OF LRRK2 ON

LYSOSOMES WITH SNX25 OR SNX27

[0001] This application claims the benefit of and priority to U.S. Application Serial No.

62/598,654 filed December 14, 2017, the entire contents of which are hereby incorporated by reference in its entirety.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

[0004] Parkinson's Disease (PD) is a degenerative disorder of the central nervous system. It results from the death of dopamine-containing cells in the substantia nigra, a region of the midbrain; the cause of cell-death is unknown. Early in the course of the disease, the most obvious symptoms are movement-related, including shaking, rigidity, slowness of movement and difficulty with walking and gait. Later, cognitive and behavioral problems may arise, with dementia commonly occurring in the advanced stages of the disease. Other symptoms include sensory, sleep and emotional problems. PD is more common in the elderly, with most cases occurring after the age of 50.

[0005] Parkinson's disease is diagnosed by a physician exam, and diagnosis is based on the medical history and a neurological examination of the patient. There is no laboratory or molecular test that will clearly identify the disease. Brain scans are sometimes used to rule out disorders that could give rise to similar symptoms. Patients may be given levodopa, or other dopamine affecting agent, and resulting relief of motor impairment tends to confirm diagnosis. The finding of Lewy bodies in the midbrain on autopsy is usually considered proof that the patient suffered from

Parkinson's disease.

[0006] Genetic factors are responsible for approximately one tenth of PD patients. Some of the most common mutations in familial PD cases affect leucine-rich repeat kinase 2 (LRRK2), a large multifunctional protein, resulting in increased kinase activity. While inhibition of LRRK2 kinase activity represents a promising disease-modifying therapy for PD, such inhibitors often cause lysosomal dysfunction that leads to toxicity.

SUMMARY OF THE INVENTION

[0007] In certain aspects, the invention provides a method of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a nucleic acid encoding SNX25, a nucleic acid encoding SNX27, or a combination thereof.

[0008] In certain aspects, the invention provides a method of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a SNX25 polypeptide, a SNX27 polypeptide, or a combination thereof.

[0009] In certain aspects, the invention provides a method of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a SNX25 activator or agonist, a SNX27 activator or agonist, or a combination thereof.

[0010] In some embodiments, the lysosomal toxicity is enlarged lysosomal puncta. In some embodiments, the LRRK2 kinase inhibitor is LRRK2-IN-1, CZC-54252, CZC-25146, HG-10-102- 1, GSK2578215A, JH-II-127, GNE-7915, GNE-0877, GNE-9605, PF-06447475, or MLi-2. In some embodiments, the subject has one or more mutations in LRRK2. In some embodiments, the one or more mutations in LRRK2 is G2019S, I2020T, R1441C/G/H, or Y1699C. In some embodiments, the subject has one or more single nucleotide polymorphisms (SNP) in LRRK2 associated with PD. In some embodiments, the subject has PD. In some embodiments, the subject is at risk of developing PD.

[0011] In certain aspects, the invention provides a method of treating PD in a subject in need thereof comprising administering to the subject a therapeutically effective amount of LRRK2 kinase inhibitor and a therapeutically effective amount of a nucleic acid encoding SNX25, a nucleic acid encoding SNX27, or a combination thereof.

[0012] In certain aspects, the invention provides a method of treating PD in a subject in need thereof comprising administering to the subject a therapeutically effective amount of LRRK2 kinase inhibitor and a therapeutically effective amount of a SNX25 polypeptide, a SNX27 polypeptide, or a combination thereof. [0013] In certain aspects, the invention provides a method of treating PD in a subject in need thereof comprising administering to the subject a therapeutically effective amount of LRRK2 kinase inhibitor and a therapeutically effective amount of a SNX25 activator or agonist, a SNX27 activator or agonist, or a combination thereof.

[0014] In some embodiments, the LRRK2 kinase inhibitor causes lysosomal toxicity. In some embodiments, the lysosomal toxicity is enlarged lysosomal puncta. In some embodiments, the

LRRK2 kinase inhibitor is LRRK2-IN-1, CZC-54252, CZC-25146, HG-10-102-1, GSK2578215A,

JH-II-127, GNE-7915, GNE-0877, GNE-9605, PF-06447475, or MLi-2. In some embodiments, the subject has one or more mutations in LRRK2. In some embodiments, the one or more mutations in

LRRK2 is G2019S, I2020T, R1441C/G/H, or Y1699C. In some embodiments, the subject has one or more single nucleotide polymorphisms (SNP) in LRRK2 associated with PD.

[0015] In certain aspects, the invention provides a method of screening for a compound that inhibits, treats, or prevents LRRK2 kinase inhibitor lysosomal toxicity, the method comprising: a) providing a cell line in cell culture; b) contacting the cells with a LRRK2 inhibitor; c) contacting the cells with a compound; d) contacting the cells with fluorescently labeled dextran; e) contacting the cells with a fluorescent plasma membrane stain; f) imaging the cells, wherein the fluorescently labeled dextran and/or the fluorescently labeled plasma membrane are imaged; g) quantifying the average size of dextran vesicles in the cells; and h) comparing the average size of dextran vesicles in the cells to the average size of dextran vesicles in cells of a control cell culture that was not contacted with the compound, wherein a reduction in the average size of dextran vesicles in the cells is indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0016] In some embodiments, the method further comprises contacting the cells with a fluorescent dye for labeling lysosomes in step d). In some embodiments, the method further comprises imaging the fluorescently labeled lysosomes in step f), quantifying the average size of lysosomal vesicles in the cells; and comparing the average size of lysosomal vesicles in the cells to the average size of lysosomal vesicles in cells of a control cell culture that was not contacted with the compound, wherein a reduction in the average size of lysosomal vesicles in the cells is further indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0017] In certain aspects, the invention provides a method of screening for a compound that inhibits, treats, or prevents LRRK2 kinase inhibitor lysosomal toxicity, the method comprising: a) providing a cell line in cell culture; b) contacting the cells with a LRRK2 inhibitor; c) contacting the cells with a compound; d) contacting the cells with a fluorescent dye for labeling lysosomes; e) contacting the cells with a fluorescent plasma membrane stain; f) imaging the cells, wherein the fluorescently labeled lysosomes and/or the fluorescently labeled plasma membrane are imaged; g) quantifying the average size of lysosomal vesicles in the cells; and h) comparing the average size of lysosomal vesicles in the cells to the average size of lysosomal vesicles in cells of a control cell culture that was not contacted with the compound, wherein a reduction in the average size of lysosomal vesicles in the cells is indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0018] In some embodiments, the method further comprises contacting the cells with a fluorescently labeled dextran in step d). In some embodiments, the method further comprises imaging the fluorescently labeled dextran in step f), quantifying the average size of dextran vesicles in the cells; and comparing the average size of dextran vesicles in the cells to the average size of dextran vesicles in cells of a control cell culture that was not contacted with the compound, wherein a reduction in the average size of dextran vesicles in the cells is further indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0019] In some embodiments, the cell line has an increased level of LRRK2. In some embodiments, CRISPR is used to increase the level of LRRK2 in the cell line. In some

embodiments, the cell line in cell culture is human motor neuron cells. In some embodiments, the cell line is cultured in a multiwell cell culture plate and wherein one or more compounds are screened, wherein each compound is applied to a separate well of the multiwell cell culture plate.

In some embodiments, the LRRK2 kinase inhibitor is LRRK2-IN-1, CZC-54252, CZC-25146, HG-

10-102-1, GSK2578215A, JH-II-127, GNE-7915, GNE-0877, GNE-9605, PF-06447475, or MLi-2.

[0020] In certain aspects, the invention provides a method of screening for a gene or protein that inhibits, treats, or prevents LRRK2 kinase inhibitor lysosomal toxicity, the method comprising: a) providing a cell line in cell culture; b) increasing or decreasing the level of a gene or protein in the cell line; c) contacting the cells with a LRRK2 inhibitor; d) contacting the cells with

fluorescently labeled dextran; e) contacting the cells with a fluorescent plasma membrane stain; f) imaging the cells, wherein the fluorescently labeled dextran and/or the fluorescently labeled plasma membrane are imaged; g) quantifying the average size of dextran vesicles in the cells; and h) comparing the average size of dextran vesicles in the cells to the average size of dextran vesicles in cells of a control cell culture wherein the level of the gene or protein was not increased or decreased, wherein a reduction in the average size of dextran vesicles in the cells is indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0021] In some embodiments, the method further comprises contacting the cells with a fluorescent dye for labeling lysosomes in step d). In some embodiments, the method further comprises imaging the fluorescently labeled lysosomes in step f), quantifying the average size of lysosomal vesicles in the cells; and comparing the average size of lysosomal vesicles in the cells to the average size of lysosomal vesicles in cells of a control cell culture wherein the level of the gene or protein was not increased or decreased, wherein a reduction in the average size of lysosomal vesicles in the cells is further indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0022] In certain aspects, the invention provides a method of screening for a gene or protein that inhibits, treats, or prevents LRRK2 kinase inhibitor lysosomal toxicity, the method comprising: a) providing a cell line in cell culture; b) increasing or decreasing the level of a gene or protein in the cell line; c) contacting the cells with a LRRK2 inhibitor; d) contacting the cells with a fluorescent dye for labeling lysosomes; e) contacting the cells with a fluorescent plasma membrane stain; f) imaging the cells, wherein the fluorescently labeled lysosomes and/or the fluorescently labeled plasma membrane are imaged; g) quantifying the average size of lysosomal vesicles in the cells; and h) comparing the average size of lysosomal vesicles in the cells to the average size of lysosomal vesicles in cells of a control cell culture wherein the level of the gene or protein was not increased or decreased, wherein a reduction in the average size of lysosomal vesicles in the cells is indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0023] In some embodiments, the method further comprises contacting the cells with a fluorescently labeled dextran in step d). In some embodiments, the method further comprises imaging the fluorescently labeled dextran in step f), quantifying the average size of dextran vesicles in the cells; and comparing the average size of dextran vesicles in the cells to the average size of dextran vesicles in cells of a control cell culture wherein the level of the gene or protein was not increased or decreased, wherein a reduction in the average size of dextran vesicles in the cells is further indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0024] In some embodiments, the cell line has an increased level of LRRK2. In some embodiments, CRISPR is used to increase the level of LRRK2 in the cell line. In some

In some embodiments, CRISPR is used to increase or decrease the level of the gene or protein in the cell line. In some embodiments, the LRRK2 kinase inhibitor is LRRK2-IN-1, CZC-54252, CZC- 25146, HG-10-102-1, GSK2578215A, JH-II-127, GNE-7915, GNE-0877, GNE-9605, PF- 06447475, or MLi-2.

BRIEF DESCRIPTION OF THE FIGURES [0025] To conform to the requirements for PCT patent applications, many of the figures presented herein are black and white representations of images originally created in color.

[0026] FIG. 1 shows the protocol used in the hMN cell assay.

[0027] FIG. 2 shows imaging of cell mask, lysotracker, and dextran in hMN cells treated with lOnm or lOOnm CZC-25146.

[0028] FIG. 3 shows CRISPR-SAM mediated activation of LRRK2.

[0029] FIG. 4 shows the efficiency of SAM tools (RT-qPCR) in SY5Y. N=3 per well.

Overexpression was specific for the target of each guide.

[0030] FIG. 5 shows the size of dextran vesicles (pm²) for control cells and cells with CRISPR- SAM activated LRRK2 with and without CZC-25146 (lOnM).

[0031] FIG. 6 shows the size of lysotracker vesicles (pm²) for control cells and cells with CRISPR-SAM activated LRRK2 with and without CZC-25146 (lOnM).

[0032] FIG. 7 shows the size of dextran vesicles (pm²) for control cells and cells with CRISPR- SAM activated LRRK2 with and without CZC-25146 (10hM and lOOnM).

[0033] FIG. 8 shows the size of lysotracker vesicles (pm²) for control cells and cells with CRISPR-SAM activated LRRK2 with and without CZC-25146 (10hM and lOOnM).

[0034] FIG. 9 shows the size of lysotracker vesicles (pm²) for control cells and cells with CRISPR-SAM activated LRRK2, SAM SNX25 and SAM SNX27 with and without CZC-25146 (lOnM and lOOnM).

[0035] FIG. 10 shows the size of dextran vesicles (pm²) for control cells and cells with

CRISPR-SAM activated LRRK2, SAM SNX25 and SAM SNX27 with and without CZC-25146 (lOnM and lOOnM).

DETAILED DESCRIPTION OF THE INVENTION

[0036] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

[0037] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.

[0038] Familial Parkinson’s Disease (PD) can be caused by mutations in the leucine-rich repeat kinase 2 (LRRK2) gene, which lead to increased LRRK2 kinase activity. Therefore, LRRK2 kinase inhibitors are attractive drug candidates for the treatment of familial PD. LRRK2 polymorphisms are also linked to increased risk of idiopathic PD. Unfortunately, some of LRRK2 inhibitors have toxic effects resulting from their interference with lysosomal activity.

[0039] Parkinson’s Disease and its genetics, evidence linking LRRK2 kinase activity to PD, pathogenic LRRK2 mutations and kinase activity, increased kinase activity and LRRK2

pathobiology, the development of potent and selective LRRK2 inhibitors and challenges facing LRRK2 inhibitors, and related topics are described in Atashrazm F, Dzamko N., LRRK2 inhibitors and their potential in the treatment of Parkinson's disease: current perspectives. Clin Pharmacol. 2016 Oct; 8: pp. 177-189, the contents of which is hereby incorporated by reference in its entirety.

[0040] Described herein is a functional relationship between LRRK2 and members of the sorting nexin (SNX) protein family. Overexpression of certain SNX proteins can protect against the deleterious lysosomal effects of LRRK2 inhibitors. Abnormal lysosomes were observed in human stem cell-derived motor neurons following treatment with an LRRK2 inhibitor, especially when LRRK2 was overexpressed using CRISPR technology. However, overexpression of SNX25 or SNX27 suppressed the inhibitor’s deleterious effect on lysosomes. As such, enhancing the activity of SNX proteins may be useful in overcoming the toxic effects of LRRK2 -targeted drugs in PD therapy and may improve the safety of these compounds in the treatment of PD

[0041] While most cases of Parkinson's Disease are idiopathic, some cases have a substantial genetic component (Cookson, MR. The role of leucine-rich repeat kinase 2 (LRRK2) in Parkinson's disease. Nat Rev Neurosci. 2010 Dec; 11(12): pp. 791-7). One of the most important genetic risk factors for Parkinson’s Disease is mutation of the leucine rich repeat kinase 2 (LRRK2) gene, which encodes a multifunctional protein that has kinase activity and protein-protein interaction domains. The exact role of LRRK2 exact role in familial Parkinson’s disease pathology is not yet well understood. Lysosomal function is disrupted in Parkinson’s disease (Henry AG,

Aghamohammadzadeh S, Samaroo H, Chen Y, Mou K, Needle E, Hirst WD. Pathogenic LRRK2 mutations, through increased kinase activity, produce enlarged lysosomes with reduced degradative capacity and increase ATP13A2 expression. Hum Mol Genet. 2015 Nov; 24(21): pp. 6013-28). Pathological LRRK2 mutations may contribute to diminished lysosomal function. Some kinase inhibitors targeting LRRK2 may be neuroprotective in Parkinson’s disease (Lee BD, Shin JH, VanKampen J, Petrucelli L, West AB, Ko HS, Lee YI, Maguire-Zeiss KA, Bowers WJ, Federoff HJ, Dawson VL, Dawson TM. Inhibitors of leucine-rich repeat kinase-2 protect against models of Parkinson's disease. Nat Med. 2010 Sep; 16(9): pp. 998-1000). LRRK2 kinase inhibitors may cause lysosomal abnormalities that lead to toxicity (Atashrazm F, Dzamko N. LRRK2 inhibitors and their potential in the treatment of Parkinson's disease: current perspectives. Clin Pharmacol. 2016 Oct; 8: pp. 177-189). Described herein is the use of CRISPR to induce expression of LRRK2 and sorting nexin (SNX) proteins in human induced pluripotent stem cell-derived motor neuron cells. Described herein is a functional relationship between certain sorting nexin proteins, SNX25 and SNX27, and LRRK2. Increased LRRK2 expression in the presence of an LRRK2 inhibitor has detrimental effect on lysosome function. Induction of SNX25 or SNX27 ameliorates the lysosomal effects of the LRRK2 inhibitor.

[0042] Parkinson’s Disease

[0043] The term "Parkinson Disease" (PD) as used herein is intended to encompass all types of Parkinson disease. In some embodiments, the term Parkinson disease means familial Parkinson disease, for example Parkinson’s disease that arises due to one or more mutations in LRRK2 such as G2019S, I2020T, R1441C/G/H, or Y1699C. In some embodiments, the term Parkinson Disease means idiopathic Parkinson Disease, or Parkinson Disease of unexplained origin: that is, Parkinson Disease that does not arise from acute exposure to toxic agents, traumatic head injury, or other external insult to the brain.

[0044] PD is the most common movement disorder of aging, and there are over a million patients who suffer from PD in the US. Pathological changes associated with PD include the loss of midbrain dopamine neurons and their axonal connections, the accumulation of intracellular aggregates composed largely of a-Synuclein (aSyn) protein, and inflammatory changes. No available therapies modify the progressive course of PD, and thus there is an urgent need for new approaches and treatment options for patients. Significant progress has been made over the past 2 decades with respect to the genetics of PD: genes have been identified as causative in familial forms of PD, and common genetic variants across the human genome have been identified that increase the risk of common non-familial PD.

[0045] Methods of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity and methods of treating Parkinson’s Disease

[0046] In certain aspects, the invention provides a method of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a nucleic acid encoding SNX25, a nucleic acid encoding SNX27, or a combination thereof.

[0047] In certain aspects, the invention provides a method of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a SNX25 polypeptide, a SNX27 polypeptide, or a combination thereof. [0048] In certain aspects, the invention provides a method of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a SNX25 activator or agonist, a SNX27 activator or agonist, or a combination thereof.

[0049] In certain aspects, the invention provides a method of inhibiting, treating, or preventing lysosomal toxicity in a subject in need thereof comprising administering to the subject a

therapeutically effective amount of a nucleic acid encoding SNX25, a nucleic acid encoding SNX27, or a combination thereof, wherein the lysosomal toxicity is caused by administration to the subject of a LRRK2 kinase inhibitor.

[0050] In certain aspects, the invention provides a method of inhibiting, treating, or preventing lysosomal toxicity in a subject in need thereof comprising administering to the subject a

therapeutically effective amount of a SNX25 polypeptide, a SNX27 polypeptide, or a combination thereof wherein the lysosomal toxicity is caused by administration to the subject of a LRRK2 kinase inhibitor.

[0051] In certain aspects, the invention provides a method of inhibiting, treating, or preventing lysosomal toxicity in a subject in need thereof comprising administering to the subject a

therapeutically effective amount of a SNX25 activator or agonist, a SNX27 activator or agonist, or a combination thereof wherein the lysosomal toxicity is caused by administration to the subject of a LRRK2 kinase inhibitor.

[0052] In some embodiments, the lysosomal toxicity is enlarged lysosomal puncta. In some embodiments, the LRRK2 kinase inhibitor is LRRK2-IN-1, CZC-54252, CZC-25146, HG-10-102- 1, GSK2578215A, JH-II-127, GNE-7915, GNE-0877, GNE-9605, PF-06447475, or MLi-2.

[0053] In some embodiments, the LRRK2 kinase inhibitor is LRRK2-IN-1 (5,l l-Dihydro-2- [[2-methoxy-4-[[4-(4-methyl-l-piperazinyl)-l-piperidinyl]carbonyl]phenyl]amino]-5,l l-dimethyl- 6H-pyrimido[4,5-b][l,4]benzodiazepin-6-one) and has the structure:

[0054] In some embodiments, the LRRK2 kinase inhibitor is CZC-54252 (N-[2-[[5-Chloro-2- [[2-methoxy-4-(4-morpholinyl)phenyl]amino]-4-pyrimidinyl]amino]phenyl]methanesulfonamide hydrochloride) and has the structure:

[0055] In some embodiments, the LRRK2 kinase inhibitor is CZC-25146 (N-[2-[[5-Fluoro-2- [[2-methoxy-4-(4-morpholinyl)phenyl]amino]-4-pyrimidinyl]amino]phenyl]methanesulfonamide) and has the structure:

[0056] In some embodiments, the LRRK2 kinase inhibitor is HG-l 0-102-1 ([4-[[5-chloro-4- (methylamino)-2-pyrimidinyl]amino]-3-methoxyphenyl]-4-morpholinyl-methanone) and has the structure:

[0057] In some embodiments, the LRRK2 kinase inhibitor is GSK2578215A (5-(2-Fluoro-4- pyridinyl)-2-(phenylmethoxy)-N-3-pyridinylbenzamide) and has the structure:

[0058] In some embodiments, the LRRK2 kinase inhibitor is JH-II-127 ([4-[[5-Chloro-4- (methylamino)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]amino]-3-methoxyphenyl]-4-morpholinyl- methanone) and has the structure:

[0059] In some embodiments, the LRRK2 kinase inhibitor is GNE-7915 ([4-[[4-(ethylamino)-5- (trifluoromethyl)-2-pyrimidinyl]amino]-2-fluoro-5-methoxyphenyl]-4-morpholinyl-methanone) and has the structure:

[0060] In some embodiments, the LRRK2 kinase inhibitor is GNE-0877 (a,a,3-trimethyl-4-[[4- (methylamino)-5-(trifluoromethyl)-2-pyrimidinyl]amino]-lH-pyrazole-l -acetonitrile) and has the structure:

[0061] In some embodiments, the LRRK2 kinase inhibitor is GNE-9605 (2-N-[5-chloro-l- [(3S,4S)-3-fluoro-l-(oxetan-3-yl)piperidin-4-yl]pyrazol-4-yl]-4-N-methyl-5- (trifluoromethyl)pyrimidine-2, 4-diamine) and has the structure:

[0062] In some embodiments, the LRRK2 kinase inhibitor is PF-06447475 (3-[4-(4- Morpholinyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]benzonitrile) and has the structure:

[0063] In some embodiments, the LRRK2 kinase inhibitor is MLi-2 (rel-3-[6-[(2R,6S)-2,6- Dimethyl-4-morpholinyl]-4-pyrimidyl]-5-[(l-methylcyclopropyl)oxy]-lH-indazole) and has the structure:

[0064] In some embodiments, the subject has one or more mutations in LRRK2. In some embodiments, the one or more mutations in LRRK2 is G2019S, I2020T, R1441C/G/H, or Y1699C. In some embodiments, the subject has one or more single nucleotide polymorphisms (SNP) in LRRK2 associated with Parkinson’s Disease. In some embodiments, the subject has Parkinson’s Disease. In some embodiments, the subject is at risk of developing Parkinson’s Disease.

[0065] In certain aspects, the invention provides a method of treating Parkinson’s Disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of LRRK2 kinase inhibitor and a therapeutically effective amount of a nucleic acid encoding SNX25, a nucleic acid encoding SNX27, or a combination thereof.

[0066] In certain aspects, the invention provides a method of treating Parkinson’s Disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of LRRK2 kinase inhibitor and a therapeutically effective amount of a SNX25 polypeptide, a SNX27 polypeptide, or a combination thereof.

[0067] In certain aspects, the invention provides a method of treating Parkinson’s Disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of LRRK2 kinase inhibitor and a therapeutically effective amount of a SNX25 activator or agonist, a SNX27 activator or agonist, or a combination thereof.

[0068] In some embodiments, the LRRK2 kinase inhibitor causes lysosomal toxicity. In some embodiments, the lysosomal toxicity is enlarged lysosomal puncta. In some embodiments, the LRRK2 kinase inhibitor is LRRK2-IN-1, CZC-54252, CZC-25146, HG-10-102-1, GSK2578215A, JH-II-127, GNE-7915, GNE-0877, GNE-9605, PF-06447475, or MLi-2. In some embodiments, the subject has one or more mutations in LRRK2. In some embodiments, the one or more mutations in LRRK2 is G2019S, I2020T, R1441C/G/H, or Y1699C. In some embodiments, the subject has one or more single nucleotide polymorphisms (SNP) in LRRK2 associated with Parkinson’s Disease.

[0069] In some embodiments, the nucleic acid encoding SNX25 or the nucleic acid encoding SNX27 is selected from the group consisting of DNA, plasmid DNA, cDNA, and mRNA.

[0070] In some embodiments, the SNX25 activator or agonist or the SNX27 activator or agonist can be molecules which, increase or prolong the activity of a protein, agonists and activators include, but are not limited to, proteins, nucleic acids, small molecules, or any other molecules which activate a protein.

[0071] Methods of screening

[0072] In certain aspects, the invention provides a method of screening for a compound that inhibits, treats, or prevents LRRK2 kinase inhibitor lysosomal toxicity, the method comprising: a) providing a cell line in cell culture; b) contacting the cells with a LRRK2 inhibitor; c) contacting the cells with a compound; d) contacting the cells with fluorescently labeled dextran; e) contacting the cells with a fluorescent plasma membrane stain; f) imaging the cells, wherein the fluorescently labeled dextran and/or the fluorescently labeled plasma membrane are imaged; g) quantifying the average size of dextran vesicles in the cells; and h) comparing the average size of dextran vesicles in the cells to the average size of dextran vesicles in cells of a control cell culture that was not contacted with the compound, wherein a reduction in the average size of dextran vesicles in the cells is indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity. [0073] In some embodiments, the method further comprises contacting the cells with a fluorescent dye for labeling lysosomes in step d). In some embodiments, the method further comprises imaging the fluorescently labeled lysosomes in step f), quantifying the average size of lysosomal vesicles in the cells; and comparing the average size of lysosomal vesicles in the cells to the average size of lysosomal vesicles in cells of a control cell culture that was not contacted with the compound, wherein a reduction in the average size of lysosomal vesicles in the cells is further indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0074] In certain aspects, the invention provides a method of screening for a compound that inhibits, treats, or prevents LRRK2 kinase inhibitor lysosomal toxicity, the method comprising: a) providing a cell line in cell culture; b) contacting the cells with a LRRK2 inhibitor; c) contacting the cells with a compound; d) contacting the cells with a fluorescent dye for labeling lysosomes; e) contacting the cells with a fluorescent plasma membrane stain; f) imaging the cells, wherein the fluorescently labeled lysosomes and/or the fluorescently labeled plasma membrane are imaged; g) quantifying the average size of lysosomal vesicles in the cells; and h) comparing the average size of lysosomal vesicles in the cells to the average size of lysosomal vesicles in cells of a control cell culture that was not contacted with the compound, wherein a reduction in the average size of lysosomal vesicles in the cells is indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0075] In some embodiments, the method further comprises contacting the cells with a fluorescently labeled dextran in step d). In some embodiments, the method further comprises imaging the fluorescently labeled dextran in step f), quantifying the average size of dextran vesicles in the cells; and comparing the average size of dextran vesicles in the cells to the average size of dextran vesicles in cells of a control cell culture that was not contacted with the compound, wherein a reduction in the average size of dextran vesicles in the cells is further indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0076] In some embodiments, the cell line has an increased level of LRRK2. In some embodiments, CRISPR is used to increase the level of LRRK2 in the cell line. In some

In some embodiments, the LRRK2 kinase inhibitor is LRRK2-IN-1, CZC-54252, CZC-25146, HG- 10-102-1, GSK2578215A, JH-II-127, GNE-7915, GNE-0877, GNE-9605, PF-06447475, or MLi-2.

[0077] In certain aspects, the invention provides a method of screening for a gene or protein that inhibits, treats, or prevents LRRK2 kinase inhibitor lysosomal toxicity, the method comprising: a) providing a cell line in cell culture; b) increasing or decreasing the level of a gene or protein in the cell line; c) contacting the cells with a LRRK2 inhibitor; d) contacting the cells with

[0078] In some embodiments, the method further comprises contacting the cells with a fluorescent dye for labeling lysosomes in step d). In some embodiments, the method further comprises imaging the fluorescently labeled lysosomes in step f), quantifying the average size of lysosomal vesicles in the cells; and comparing the average size of lysosomal vesicles in the cells to the average size of lysosomal vesicles in cells of a control cell culture wherein the level of the gene or protein was not increased or decreased, wherein a reduction in the average size of lysosomal vesicles in the cells is further indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0079] In certain aspects, the invention provides a method of screening for a gene or protein that inhibits, treats, or prevents LRRK2 kinase inhibitor lysosomal toxicity, the method comprising: a) providing a cell line in cell culture; b) increasing or decreasing the level of a gene or protein in the cell line; c) contacting the cells with a LRRK2 inhibitor; d) contacting the cells with a fluorescent dye for labeling lysosomes; e) contacting the cells with a fluorescent plasma membrane stain; f) imaging the cells, wherein the fluorescently labeled lysosomes and/or the fluorescently labeled plasma membrane are imaged; g) quantifying the average size of lysosomal vesicles in the cells; and h) comparing the average size of lysosomal vesicles in the cells to the average size of lysosomal vesicles in cells of a control cell culture wherein the level of the gene or protein was not increased or decreased, wherein a reduction in the average size of lysosomal vesicles in the cells is indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0080] In some embodiments, the method further comprises contacting the cells with a fluorescently labeled dextran in step d). In some embodiments, the method further comprises imaging the fluorescently labeled dextran in step f), quantifying the average size of dextran vesicles in the cells; and comparing the average size of dextran vesicles in the cells to the average size of dextran vesicles in cells of a control cell culture wherein the level of the gene or protein was not increased or decreased, wherein a reduction in the average size of dextran vesicles in the cells is further indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

[0081] In some embodiments, the cell line has an increased level of LRRK2. In some embodiments, CRISPR is used to increase the level of LRRK2 in the cell line. In some

[0082] As used herein, a compound can be, but is not limited to, a compound that inhibits, treats, prevents or reduces lysosomal toxicity caused by LRRK2 kinase. As used herein, a compound can also be, but is not limited to, a compound that interacts with a gene, or protein, polypeptide, or peptide, and modulates its activity or its expression. Some non-limiting examples of compounds include peptides (such as peptide fragments comprising a polypeptide encoded by a gene, or antibodies or fragments thereof), small molecules, and nucleic acids (such as siRNA or antisense RNA specific for a nucleic acid). The compound can either increase the activity or expression of a protein encoded by a gene, or the compound can decrease the activity or expression of a protein encoded by a gene.

[0083] The compound can be an antagonist (e.g., an inhibitor). Antagonists can be molecules which, decrease the amount or the duration of the activity of a protein. Antagonists and inhibitors include proteins, nucleic acids, antibodies, small molecules, or any other molecules which decrease the activity of a protein.

[0084] The compound can be an agonist. Agonists of a protein can be molecules which, increase or prolong the activity of a protein, agonists include, but are not limited to, proteins, nucleic acids, small molecules, or any other molecules which activate a protein.

[0085] The methods can comprise the identification of test compounds or agents (e.g., peptides (such as antibodies or fragments thereof), small molecules, nucleic acids (such as siRNA or antisense RNA), or other agents) that can inhibit, treat, prevent or reduce lysosomal toxicity caused by LRRK2 kinase inhibitors.

[0086] In one embodiment, a compound can be a peptide fragment. Fragments include all possible amino acid lengths between and including about 8 and about 100 amino acids, for example, lengths between about 10 and about 100 amino acids, between about 15 and about 100 amino acids, between about 20 and about 100 amino acids, between about 35 and about 100 amino acids, between about 40 and about 100 amino acids, between about 50 and about 100 amino acids, between about 70 and about 100 amino acids, between about 75 and about 100 amino acids, or between about 80 and about 100 amino acids. These peptide fragments can be obtained

commercially or synthesized via liquid phase or solid phase synthesis methods (Atherton et al. , (1989) Solid Phase Peptide Synthesis: a Practical Approach. IRL Press, Oxford, England). The peptide fragments can be isolated from a natural source, genetically engineered, or chemically prepared. These methods are well known in the art.

[0087] A compound, for example, an agonist or antagonist, can be a protein such as an antibody (monoclonal, polyclonal, humanized, chimeric, or fully human), or a binding fragment thereof. An antibody fragment can be a form of an antibody other than the full-length form and includes portions or components that exist within full-length antibodies, in addition to antibody fragments that have been engineered. Antibody fragments can include, but are not limited to, single chain Fv (scFv), diabodies, Fv, and (Fab')2, triabodies, Fc, Fab, CDR1, CDR2, CDR3, combinations of CDR’s, variable regions, tetrabodies, bifunctional hybrid antibodies, framework regions, constant regions, and the like (see, Maynard et a/., (2000) Ann. Rev. Biomed. Eng. 2:339-76; Hudson (1998) Curr. Opin. Biotechnol. 9:395-402). Antibodies can be obtained commercially, custom generated, or synthesized against an antigen of interest according to methods established in the art (Janeway et al, (2001) Immunobiology, 5th ed., Garland Publishing).

[0088] A compound, for example, an agonist or antagonist, can be selected from the group comprising: siRNA; interfering RNA or RNAi; dsRNA; RNA Polymerase III transcribed DNAs; ribozymes; and antisense nucleic acids, which can be RNA, DNA, or an artificial nucleic acid. Antisense oligonucleotides, including antisense DNA, RNA, and DNA/RNA molecules, act to directly block the translation of mRNA by binding to targeted mRNA, and preventing protein translation. Antisense oligonucleotides of at least about 15 bases can be synthesized, e.g., by conventional phosphodiester techniques (Dallas et al, (2006) Med. Sci. Monit. l2(4):RA67-74; Kalota et al, (2006) Handb. Exp. Pharmacol. 173: 173-96; Lutzel burger et al, (2006) Handb. Exp. Pharmacol. 173:243-59). Antisense nucleotide sequences include, but are not limited to:

morpholinos, 2’-0-methyl polynucleotides, DNA, RNA and the like.

[0089] siRNA comprises a double stranded structure containing from about 15 to about 50 base pairs, for example from about 21 to about 25 base pairs, and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. siRNA comprises a sense

RNA strand and a complementary antisense RNA strand annealed together by standard Watson-

Crick base-pairing interactions. The sense strand comprises a nucleic acid sequence which is substantially identical to a nucleic acid sequence contained within the target miRNA molecule. “Substantially identical” to a target sequence contained within the target mRNA refers to a nucleic acid sequence that differs from the target sequence by about 3% or less. The sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules, or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded“hairpin” area. See also , McManus and Sharp (2002) Nat Rev Genetics , 3:737-47, and Sen and Blau (2006) FASEB J , 20: 1293-99, the entire disclosures of which are herein incorporated by reference.

[0090] The siRNA can be altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxyribonucleotides. One or both strands of the siRNA can also comprise a 3’ overhang. As used herein, a 3’ overhang refers to at least one unpaired nucleotide extending from the 3’ -end of a duplexed RNA strand. For example, the siRNA can comprise at least one 3’ overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxyribonucleotides) in length, or from 1 to about 5 nucleotides in length, or from 1 to about 4 nucleotides in length, or from about 2 to about 4 nucleotides in length. For example, each strand of the siRNA can comprise 3’ overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).

[0091] siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector (for example, see U.S. Patent No. 7,294,504 and U.S. Patent No. 7,422,896, the entire disclosures of which are herein incorporated by reference). Exemplary methods for producing and testing dsRNA or siRNA molecules are described in ET.S. Patent Application Publication No. 2002/0173478 to Gewirtz, ET.S. Patent Application Publication No. 2007/0072204 to Hannon et al ., and in U.S. Patent Application Publication No.2004/0018176 to Reich et al. , the entire disclosures of which are herein incorporated by reference.

[0092] RNA polymerase III transcribed DNAs contain promoters, such as the U6 promoter. These DNAs can be transcribed to produce small hairpin RNAs in the cell that can function as siRNA or linear RNAs that can function as antisense RNA. A compound, for example, an agonist or antagonist, can contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited. In addition, these forms of nucleic acid can be single, double, triple, or quadruple stranded (see for example Bass (2001)

Nature , 411, 428 429; Elbashir et al. , (2001) Nature , 411, 494 498; and PCT Publication Nos. WO 00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO 01/29058, WO 99/07409, WO 00/44914).

[0093] A compound, for example, an agonist or antagonist, can be a small molecule that binds to a protein and disrupts its function, or conversely, enhances its function. Small molecules are a diverse group of synthetic and natural substances generally having low molecular weights. They can be isolated from natural sources (for example, plants, fungi, microbes and the like), are obtained commercially and/or available as libraries or collections, or synthesized. Candidate small molecules can be identified via in silico screening or high-through-put (HTP) screening of combinatorial libraries. Most conventional pharmaceuticals, such as aspirin, penicillin, and many chemotherapeutics, are small molecules, can be obtained commercially, can be chemically synthesized, or can be obtained from random or combinatorial libraries as described below (Werner et al. , (2006) Brief Fund. Genomic Proteomic 5(1): 32-6).

[0094] Knowledge of the primary sequence of a molecule of interest, such as the amino acid sequence, and the similarity of that sequence with proteins of known function, can provide information as to the inhibitors or antagonists of the protein of interest in addition to agonists.

Identification and screening of agonists and antagonists is further facilitated by determining structural features of the protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide for the rational design or identification of agonists and antagonists.

[0095] Test compounds, for example, an agonist or antagonist, can be screened from large libraries of synthetic or natural compounds (see Wang et al. , (2007) Curr Med Chem , 14(2): 133-55; Mannhold (2006) Curr Top Med Chem , 6 (10): 1031-47; and Hensen (2006) Curr Med Chem l3(4):36l-76). Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available. Rare chemical libraries are also available, as well libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts which are also readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al. , (1996) Tib Tech 14:60).

[0096] Methods for preparing libraries of molecules are well known in the art and many libraries are commercially available. Libraries of interest in the invention include peptide libraries, randomized oligonucleotide libraries, synthetic organic combinatorial libraries, and the like.

Degenerate peptide libraries can be readily prepared in solution, in immobilized form as bacterial flagella peptide display libraries or as phage display libraries. Peptide ligands can be selected from combinatorial libraries of peptides containing at least one amino acid. Libraries can be synthesized of peptides and non-peptide synthetic moieties. Such libraries can further be synthesized which contain non-peptide synthetic moieties, which are less subject to enzymatic degradation compared to their naturally-occurring counterparts. For example, libraries can also include, but are not limited to, peptide-on-plasmid libraries, synthetic small molecule libraries, aptamer libraries, in vitro translation-based libraries, polysome libraries, synthetic peptide libraries, neurotransmitter libraries, and chemical libraries.

[0097] Small molecule combinatorial libraries can also be generated and screened. A combinatorial library of small organic compounds is a collection of closely related analogs that differ from each other in one or more points of diversity and are synthesized by organic techniques using multi-step processes. Combinatorial libraries include a vast number of small organic compounds. One type of combinatorial library is prepared by means of parallel synthesis methods to produce a compound array. A compound array can be a collection of compounds identifiable by their spatial addresses in Cartesian coordinates and arranged such that each compound has a common molecular core and one or more variable structural diversity elements. The compounds in such a compound array are produced in parallel in separate reaction vessels, with each compound identified and tracked by its spatial address. Examples of parallel synthesis mixtures and parallel synthesis methods are provided in U.S. Ser. No. 08/177,497, filed Jan. 5, 1994 and its

corresponding PCT Publication No. WO 95/18972, published Jul. 13, 1995 and U.S. Pat. No.

5,712,171 granted Jan. 27, 1998 and its corresponding PCT Publication No. WO 96/22529, which are hereby incorporated by reference.

[0098] In one non-limiting example, non-peptide libraries, such as a benzodiazepine library (see e.g., Bunin et al., (1994 ) Proc. Natl. Acad. Sci. USA 91 :4708-4712), can be screened. Peptoid libraries, such as that described by Simon et al., (1992) Proc. Natl. Acad. Sci. USA 89:9367-9371, can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994), Proc. Natl. Acad. Sci. USA 91 : 11138-11142.

[0099] Nucleotide-based Compounds: In one aspect, a compound of the invention is a nucleotide-based agonist or antagonist, inhibitor or activator, of a gene. Such inhibitors or antagonists include, but are not limited to siRNAs, shRNAs, dsRNAs, microRNAs, antisense RNA molecules, and ribozymes, that inhibit the expression or activity of a gene. Such nucleotide-based inhibitors may comprise ribonucleotides, deoxyribonucleotides, or various artificial nucleotide derivatives. [0100] siRNA: RNA interference (RNAi) is a method of gene-specific silencing which employs sequence-specific small interfering RNA (siRNA) to target and degrade the gene-specific mRNA prior to translation. Methods for designing specific siRNAs based on an mRNA sequence are well known in the art and design algorithms are available on the websites of many commercial vendors that synthesize siRNAs, including Dharmacon, Ambion, Qiagen, GenScript and Clontech.

[0101] Antisense: Antisense oligonucleotides (ASOs) are small deoxy-oligonucleotides with a sequence complementary to the mRNA of the target gene (Crooke, (1993) Curr. Opin. Invest.

Drugs , 2: 1045-1048; Stein and Cheng, (1993) Science , 261 : 1004-10012; Hawley and Gibson (1996) Antisense & Nucleic Drug Dev ., 6: 185-195; Crooke, S.T. (2003) Ann. Rev. Med., 55: 61-95; Kalota, et ah, (2004) Cancer Biol. & Therapy , 3 : 4-12; Orr, et ah, (2005) Meth. Mol. Med., 106: 85- 111). They bind to the target mRNA through complementary base-pairing and attract the binding of RNase H, an enzyme that degrades double strand RNA, thus destroying the target mRNA (18- 25). While unmodified ASOs can be as sensitive to degradation as RNA, chemical modification of the phosphodiester backbones can make them resistant to the degradative action of nucleases in in vivo situations (nonlimiting examples include phosphorothioate- or 2’-0-[2-methoxyethyl]- backbone modifications) (Monia, et al. (1996) J. Biol. Chem., 271 : 14533-1440; also see U.S.

Patent Nos. 5,652,355 and 5,652,356).

[0102] ASOs offer many unique aspects including: 1) they are simple defined chemical agents can be synthesized in bulk under highly controlled (good clinical practice) conditions; 2) they can be delivered to patients systemically in controlled doses, making it more likely that they can even reach distal metastases; 3) they are not known to have potential for genetic damage, as with other biological agents (viruses) that are being developed and tested for gene therapy strategies and; 4) gene-targeting ASO agents are already in clinical trials for several different cancers, thus there already is a body of literature regarding their use in humans.

[0103] shRNAs: Small interfering RNAs can be expressed in vivo in the form of short, fold- back, hairpin loop structures known as short hairpin RNAs (shRNAs) comprising the siRNA sequence of interest. When expressed in a cell, shRNA is rapidly processed by intracellular machinery into siRNA. Expression of shRNAs is accomplished by ligating the shRNA into an expression cassette of a double stranded RNA (dsRNA) expression vector. Expression may be driven by RNA polymerase III promoters (See ET.S. Patent No. 6,852,535). Plasmid vectors for expression of shRNAs are commercially available from vendors such as Gene Therapy Systems,

Ambion and Stratagene. ET.S. Publication No. ETS2005/0019918A1 describes the use of a lentiviral vector for in vivo siRNA expression. Methods for DNA and RNA manipulations, including ligation and purification, are well known to those skilled in the art. Vectors comprising shRNA expression cassettes may be introduced into prokaryotic or eukaryotic cells using methods known to one skilled in the art.

[0104] Short hairpin RNAs are available through commercial vendors, many vendors also have online algorithms useful for designing shRNAs (i.e., Clontech, ExpressOn, Gene Link and BD Biosciences).

[0105] PNA: Peptide nucleic acids (PNAs) comprise naturally-occurring DNA bases (i.e., adenine, thymine, cytosine, guanine) or artificial bases (i.e., bromothymine, azaadenines, azaguanines) attached to a peptide backbone through a suitable linker. Nonlimiting examples of PNA backbone linking moieties include amide, thioamide, sulfmamide or sulfonamide linkages. Preferably, the linking moieties in the PNA backbone comprise N-ethylaminoglycine units, and the bases are covalently bound to the PNA backbone by methylene-carbonyl groups. PNAs bind complementary DNA or RNA strands more strongly than a corresponding DNA. They can be utilized in a manner similar to antisense oligonucleotides to block the translation of specific mRNA transcripts. PNA oligomers can be prepared according to the method provided by U.S. Patent No. 6,713,602. U.S. Patent No. 6,723,560 describes methods for modulating transcription and translation using sense and antisense PNA oligomers, respectively. Also included in this patent are methods for administration of PNAs to a subject such that the oligomers cross biological barriers and engender a sequence specific response. The PNA can be attached to a targeting moiety, such as an internalization peptide, facilitate uptake of the PNA by cells or tissues.

[0106] Peptides and Peptidomimetics: Proteins and peptides may be synthesized by methods well known in the art, including chemical synthesis and recombinant DNA methods. A

peptidomimetic is a compound that is structurally similar to a peptide, such that the peptidomimetic retains the functional characteristics of the peptide. Peptidomimetics include organic compounds and modified peptides that mimic the three-dimensional shape of a peptide. As described in U.S. Patent No. 5,331,573, the shape of the peptidomimetic may be designed and evaluated using techniques such as NMR or computational techniques. Inhibitors or activators can be designed based on the structural characteristics of the proteins of interest. Mutational analyses known in the art may be used to define amino acids or amino acid sequences required for protein-protein interactions.

[0107] Within the scope of the present invention are peptide or peptidomimetic inhibitors and activators sharing sufficient homology with and binding to the interaction domains, or portions thereof, which may be used, for example, to block complex formation between a protein and its receptors, or enhance the activity of a protein. [0108] The invention encompasses a composition comprising one or more proteins or peptides provided for by the invention and a pharmaceutically acceptable carrier. The invention also encompasses a composition comprising one or more peptidomimetics provided for by the invention and a pharmaceutically acceptable carrier.

[0109] In one embodiment, an inhibitor, or an activator, can be a peptide fragment. For example, the fragment can encompass any portion of at least about 8 consecutive amino acids. The fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, at least about 50 consecutive amino acids, at least about 60 consecutive amino acids, or at least about 75 consecutive amino acids. Fragments include all possible amino acid lengths between and including about 8 and about 100 amino acids, for example, lengths between about 10 and about 100 amino acids, between about 15 and about 100 amino acids, between about 20 and about 100 amino acids, between about 35 and about 100 amino acids, between about 40 and about 100 amino acids, between about 50 and about 100 amino acids, between about 70 and about 100 amino acids, between about 75 and about 100 amino acids, or between about 80 and about 100 amino acids.

These peptide fragments can be obtained commercially or synthesized via liquid phase or solid phase synthesis methods (Atherton et al., (1989) Solid Phase Peptide Synthesis: A Practical Approach. IRL Press, Oxford, England).

[0110] Antibodies: In one aspect, a compound of the invention is an antibody agonist or antagonist, inhibitor or activator, or a fragment thereof. The invention provides for an antibody, or antigen-binding fragment thereof, that specifically binds to a protein. Within the context of the invention, the antibody, or fragment thereof, can be monoclonal, polyclonal, chimeric or humanized. Such antibodies and antigen-binding fragments may be used, for example, to block complex formation between a protein and its receptors, or enhance the activity of a protein, or modify its interaction with lipids or cholesterol.

[0111] A compound of the invention can be a protein, such as an antibody (monoclonal, polyclonal, humanized, chimeric, or fully human), or a binding fragment thereof, directed against a polypeptide. An antibody fragment can be a form of an antibody other than the full-length form and includes portions or components that exist within full-length antibodies, in addition to antibody fragments that have been engineered. Antibody fragments can include, but are not limited to, single chain Fv (scFv), diabodies, Fv, and (Fab')2, triabodies, Fc, Fab, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, tetrabodies, bifunctional hybrid antibodies, framework regions, constant regions, and the like (see, Maynard et al., (2000) Ann. Rev. Biomed. Eng. 2:339-76; Hudson (1998)

Curr. Opin. Biotechnol. 9:395-402). Antibodies can be obtained commercially, custom generated, or synthesized against an antigen of interest according to methods established in the art (Janeway et al ., (2001) Immunobiology, 5th ed., Garland Publishing).

[0112] Small molecules: Within the scope of the invention, the small molecule comprises an organic molecule. Also within the scope of the invention, the small molecule comprises an inorganic molecule. Protein-protein interaction inhibitors may act directly via inhibition at the protein-protein interface, or indirectly via binding to a site not at the interface and inducing a conformational change in the protein such that the protein is prohibited from engaging in the protein-protein interaction (Pagliaro et al, Curr Opin Chem Biol 8:442-449 (2004)). U.S.

Publication No. US 2005/0032245A1 describes methods for determining such inhibitors and evaluating potential inhibitors that prevent or inhibit protein-protein interactions.

[0113] A compound of the invention can also be a small molecule that binds to a protein and disrupts its function. Small molecules are a diverse group of synthetic and natural substances generally having low molecular weights. They can be isolated from natural sources (for example, plants, fungi, microbes and the like), are obtained commercially and/or available as libraries or collections, or synthesized. Candidate small molecules that modulate a protein can be identified via in silico screening or high-through-put (HTP) screening of combinatorial libraries. Most conventional pharmaceuticals, such as aspirin, penicillin, and many chemotherapeutics, are small molecules, can be obtained commercially, can be chemically synthesized, or can be obtained from random or combinatorial libraries (Werner et al., (2006) Brief Funct. Genomic Proteomic 5(l):32- 6). In some embodiments, the agent is a small molecule that binds, interacts, or associates with a target protein or RNA. Such a small molecule can be an organic molecule that, when the target is an intracellular target, is capable of penetrating the lipid bilayer of a cell to interact with the target. Small molecules include, but are not limited to, toxins, chelating agents, metals, and metalloid compounds. Small molecules can be attached or conjugated to a targeting agent so as to specifically guide the small molecule to a particular cell.

[0114] Additional examples for determining inhibitors or antagonists, activators or agonists of use the protein crystal structure. The crystal structure may be used to screen for inhibitors or antagonists, activators or agonists or to design inhibitors or antagonists, activators or agonists, respectively. One of ordinary skill in the art can solve the crystal structure of and determine sites which confer function, respectively. Based on the crystal structure, in silico screens of compound databases may be performed to discover compounds that would be predicted to inhibit or activate proteins. These compounds can then be evaluated in assays to determine if they inhibit or activate protein function. Additionally, the crystal structure can be used to design compounds (i.e., rational drug design) that would be predicted to inhibit or activate protein function based on the structure of the compound, then the compound can be tested in assays to determine if they inhibit or activate protein function.

[0115] One of skill in the art will understand that other agents may be useful as agonist or antagonist, inhibitor or activators and may be used in conjunction with the methods of the invention.

[0116] DNA and Amino Acid Manipulation Methods and Purification Thereof

[0117] The present invention utilizes conventional molecular biology, microbiology, and recombinant DNA techniques available to one of ordinary skill in the art. Such techniques are well known to the skilled worker and are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual" (1982): "DNA Cloning: A Practical Approach," Volumes I and II (D. N. Glover, ed., 1985); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Nucleic Acid Hybridization" (B. D. Hames & S. J. Higgins, eds., 1985); "Transcription and Translation" (B. D. Hames & S. J. Higgins, eds., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1986); "Immobilized Cells and Enzymes" (IRL Press, 1986): B. Perbal, "A Practical Guide to Molecular Cloning" (1984), and Sambrook, et ak, "Molecular Cloning: a Laboratory Manual" (1989).

[0118] One skilled in the art can obtain a protein in several ways, which include, but are not limited to, isolating the protein via biochemical means or expressing a nucleotide sequence encoding the protein of interest by genetic engineering methods.

[0119] A protein is encoded by a nucleic acid (including, for example, genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA). For example, it can be encoded by a recombinant nucleic acid of a gene. The proteins and nucleic acids of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a protein can be obtained by screening DNA libraries, or by amplification from a natural source. A protein can be a fragment or portion thereof. The nucleic acids encoding a protein can be produced via recombinant DNA technology and such recombinant nucleic acids can be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof.

[0120] The abbreviation“SNX25” refers to sorting nexin 25 (NCBI gene ID: 83891). The nucleic acid sequences of the genes encoding SNX25, including, but not limited to, the nucleic acid sequences of the open reading frames of the genes, are known in the art. The amino acid sequences of SNX25 polypeptides and proteins, including, but not limited to, the amino acid sequences of the human SNX25 polypeptides and proteins are known in the art. Reference herein to the SNX25 protein encompasses reference to any processed form of the protein. Sequence information related to the SNX 25 proteins and nucleic acids of the invention are accessible in public databases, such as GenBank. The genomic DNA sequence of SNX25 is GenBank number NC_000004.12 and is hereby incorporated by reference in its entirety. The mRNA and cDNA sequences of SNX25 is GenBank number NM 001317781.1 which is hereby incorporated by reference in its entirety. The protein sequence of SNX25 is GenBank number NR_001304710.1 which is hereby incorporated by reference in its entirety.

[0121] The abbreviation“SNX27” refers to sorting nexin 27 (NCBI gene ID: 81609). The nucleic acid sequences of the genes encoding SNX27, including, but not limited to, the nucleic acid sequences of the open reading frames of the genes, are known in the art. The amino acid sequences of SNX27 polypeptides and proteins, including, but not limited to, the amino acid sequences of the human SNX27 polypeptides and proteins are known in the art. Reference herein to the SNX27 protein encompasses reference to any processed form of the protein. Sequence information related to the SNX 27 proteins and nucleic acids of the invention are accessible in public databases, such as GenBank. The gene encodes two transcript variants (NM_001330723.1; NM_0309l8.5).

Transcript variant 1 (NM_001330723.1) encodes isoform 1 (NP_00l317652.1). Transcript variant 2 (NM 030918.5) differs in its 3’UTR and coding sequence compared to transcript variant 2 and encodes isoform 2 (NP_l 12180.4) which has a shorter and distinct C-terminus compared to isoform 1. The genomic DNA sequence of SNX27 is GenBank number NC_00000l .11 and is hereby incorporated by reference in its entirety. The mRNA and cDNA sequences of SNX27 is GenBank numbers NM_001330723.1 and NM_0309l8.5 which are hereby incorporated by reference in their entireties. The protein sequence of SNX27 is GenBank numbers NP_00l317652.1 and

NP_l 12180.4 which are hereby incorporated by reference in their entireties.

[0122] The proteins of the invention (e.g., SNX25, SNX27) are encoded by a nucleic acid (including, for example, genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA). For example, they can be encoded by a recombinant nucleic acid of their corresponding gene. The proteins of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a protein can be obtained by screening DNA libraries, or by amplification from a natural source. A protein can be a fragment or portion thereof. The nucleic acids encoding a protein can be produced via recombinant DNA technology and such recombinant nucleic acids can be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof. Sequence information related to the proteins and nucleic acids of the invention are accessible in public databases, such as GenBank. [0123] Protein Variants: Protein variants can include amino acid sequence modifications. For example, amino acid sequence modifications fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions can include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. These variants ordinarily are prepared by site-specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.

[0124] Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions can be single residues, but can occur at a number of different locations at once. In one non-limiting embodiment, insertions can be on the order of about from 1 to about 10 amino acid residues, while deletions can range from about 1 to about 30 residues. Deletions or insertions can be made in adjacent pairs (for example, a deletion of about 2 residues or insertion of about 2 residues). Substitutions, deletions, insertions, or any combination thereof can be combined to arrive at a final construct. The mutations cannot place the sequence out of reading frame and should not create complementary regions that can produce secondary mRNA structure.

Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place.

[0125] Substantial changes in function are made by selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions that can produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,

phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue;

(c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for

(or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and/or glycosylation.

[0126] Minor variations in the amino acid sequences of proteins are provided by the present invention. The variations in the amino acid sequence can be when the sequence maintains at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% identity to the wild type sequence. For example, conservative amino acid replacements can be utilized. Conservative replacements are those that take place within a family of amino acids that are related in their side chains, wherein the interchangeability of residues have similar side chains.

[0127] Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) a group of amino acids having aliphatic-hydroxyl side chains, such as serine and threonine; (ii) a group of amino acids having amide-containing side chains, such as asparagine and glutamine; (iii) a group of amino acids having aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; (iv) a group of amino acids having aromatic side chains, such as phenylalanine, tyrosine, and tryptophan; and (v) a group of amino acids having sulfur-containing side chains, such as cysteine and methionine. Useful conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic-aspartic, and asparagine-glutamine.

[0128] For example, the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, Gly,

Ala; Val, Ile, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. Substitutional or deletional mutagenesis can be employed to insert sites for N-glycosylation (Asn-X-Thr/Ser) or O- glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also can be desirable.

Deletions or substitutions of potential proteolysis sites, e.g. Arg, is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.

[0129] Bacterial and Yeast Expression Systems: In bacterial systems, a number of expression vectors can be selected. For example, when a large quantity of a protein encoded by a gene is needed, vectors which direct high level expression of proteins that are readily purified can be used.

Non-limiting examples of such vectors include multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). pIN vectors or pGEX vectors (Promega, Madison,

Wis.) also can be used to express foreign polypeptide molecules as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

[0130] Plant and Insect Expression Systems: If plant expression vectors are used, the expression of sequences encoding a protein can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV. Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters, can be used. These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.

[0131] An insect system also can be used to express proteins. For example, in one such system Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. Sequences encoding a polypeptide can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of nucleic acid sequences, such as a sequence corresponding to a gene will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which the protein or a variant thereof can be expressed.

[0132] Mammalian Expression Systems: An expression vector can include a nucleotide sequence that encodes a polypeptide linked to at least one regulatory sequence in a manner allowing expression of the nucleotide sequence in a host cell. A number of viral -based expression systems can be used to express a protein or a variant thereof in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding a protein can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion into a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which expresses a protein in infected host cells. Transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can also be used to increase expression in mammalian host cells.

[0133] Regulatory sequences are well known in the art, and can be selected to direct the expression of a protein or polypeptide of interest in an appropriate host cell as described in

Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego,

Calif. (1990). Non-limiting examples of regulatory sequences include: polyadenylation signals, promoters (such as CMV, ASV, SV40, or other viral promoters such as those derived from bovine papilloma, polyoma, and Adenovirus 2 viruses (Fiers, et al., 1973, Nature 273: 113; Hager GL, et al., Curr Opin Genet Dev, 2002, 12(2): 137-41) enhancers, and other expression control elements.

[0134] Enhancer regions, which are those sequences found upstream or downstream of the promoter region in non-coding DNA regions, are also known in the art to be important in optimizing expression. If needed, origins of replication from viral sources can be employed, such as if a prokaryotic host is utilized for introduction of plasmid DNA. However, in eukaryotic organisms, chromosome integration is a common mechanism for DNA replication.

[0135] For stable transfection of mammalian cells, a small fraction of cells can integrate introduced DNA into their genomes. The expression vector and transfection method utilized can be factors that contribute to a successful integration event. For stable amplification and expression of a desired protein, a vector containing DNA encoding a protein of interest is stably integrated into the genome of eukaryotic cells (for example mammalian cells, such as cells from the end bulb of the hair follicle), resulting in the stable expression of transfected genes. An exogenous nucleic acid sequence can be introduced into a cell (such as a mammalian cell, either a primary or secondary cell) by homologous recombination as disclosed in U.S. Patent 5,641,670, the contents of which are herein incorporated by reference.

[0136] A gene that encodes a selectable marker (for example, resistance to antibiotics or drugs, such as ampicillin, neomycin, G418, and hygromycin) can be introduced into host cells along with the gene of interest in order to identify and select clones that stably express a gene encoding a protein of interest. The gene encoding a selectable marker can be introduced into a host cell on the same plasmid as the gene of interest or can be introduced on a separate plasmid. Cells containing the gene of interest can be identified by drug selection wherein cells that have incorporated the selectable marker gene will survive in the presence of the drug. Cells that have not incorporated the gene for the selectable marker die. Surviving cells can then be screened for the production of the desired protein molecule.

[0137] Cell Transfection

[0138] A eukaryotic expression vector can be used to transfect cells in order to produce proteins encoded by nucleotide sequences of the vector. Mammalian cells, such as neuronal cells or brain tissue, can contain an expression vector via introducing the expression vector into an appropriate host cell via methods known in the art.

[0139] A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed polypeptide encoded by a gene in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a“prepro” form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, Va. 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.

[0140] An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art, such as lipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextran-mediated transfection, or electroporation. Electroporation is carried out at approximate voltage and capacitance to result in entry of the DNA construct(s) into cells of interest (such as neuronal cells and brain cells). Other transfection methods also include modified calcium phosphate precipitation, polybrene precipitation, liposome fusion, and receptor-mediated gene delivery.

[0141] Cells that will be genetically engineered can be primary and secondary cells obtained from various tissues, and include cell types which can be maintained and propagated in culture. Non-limiting examples of primary and secondary cells include epithelial cells, neuronal cells, brain cells, endothelial cells, glial cells, fibroblasts, muscle cells (such as myoblasts) keratinocytes, formed elements of the blood (e.g., lymphocytes, bone marrow cells), and precursors of these somatic cell types.

[0142] Vertebrate tissue can be obtained by methods known to one skilled in the art, such a punch biopsy or other surgical methods of obtaining a tissue source of the primary cell type of interest. In one embodiment, a punch biopsy or removal can be used to obtain a source of keratinocytes, fibroblasts, endothelial cells, or mesenchymal cells. In another embodiment, removal of a hair follicle can be used to obtain a source of fibroblasts, keratinocytes, endothelial cells, or mesenchymal cells. A mixture of primary cells can be obtained from the tissue, using methods readily practiced in the art, such as explanting or enzymatic digestion (for examples using enzymes such as pronase, trypsin, collagenase, elastase dispase, and chymotrypsin). Biopsy methods have also been described in United States Patent Application Publication 2004/0057937 and PCT application publication WO 2001/32840, and are hereby incorporated by reference.

[0143] Primary cells can be acquired from the individual to whom the genetically engineered primary or secondary cells are administered. However, primary cells can also be obtained from a donor, other than the recipient, of the same species. The cells can also be obtained from another species (for example, rabbit, cat, mouse, rat, sheep, goat, dog, horse, cow, bird, or pig). Primary cells can also include cells from an isolated vertebrate tissue source grown attached to a tissue culture substrate (for example, flask or dish) or grown in a suspension; cells present in an explant derived from tissue; both of the aforementioned cell types plated for the first time; and cell culture suspensions derived from these plated cells. Secondary cells can be plated primary cells that are removed from the culture substrate and replated, or passaged, in addition to cells from the subsequent passages. Secondary cells can be passaged one or more times. These primary or secondary cells can contain expression vectors having a gene that encodes a protein of interest.

[0144] Cell Culturing

[0145] Various culturing parameters can be used with respect to the host cell being cultured. Appropriate culture conditions for mammalian cells are well known in the art (Cleveland WL, et ah, J Immunol Methods, 1983, 56(2): 221-234) or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. (Oxford University Press: New York, 1992), which is incorporated herein by reference in its entirety). Cell culturing conditions can vary according to the type of host cell selected.

Commercially available medium can be utilized. Non-limiting examples of medium include, for example, Minimal Essential Medium (MEM, Sigma, St. Louis, Mo.); Dulbecco's Modified Eagles Medium (DMEM, Sigma); Ham's F10 Medium (Sigma); HyClone cell culture medium (HyClone, Logan, Utah); RPMI-1640 Medium (Sigma); and chemically-defined (CD) media, which are formulated for various cell types, e.g., CD-CHO Medium (Invitrogen, Carlsbad, Calif.).

[0146] The cell culture media can be supplemented as necessary with supplementary

components or ingredients, including optional components, in appropriate concentrations or amounts, as necessary or desired. Cell culture medium solutions provide at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbohydrate such as glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; (3) vitamins and/or other organic compounds required at low

concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that can be required at very low concentrations, usually in the micromolar range.

[0147] The medium also can be supplemented electively with one or more components from any of the following categories: (1) salts, for example, magnesium, calcium, and phosphate; (2) hormones and other growth factors such as, serum, insulin, transferrin, and epidermal growth factor; (3) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (4) nucleosides and bases such as, adenosine, thymidine, and hypoxanthine; (5) buffers, such as HEPES; (6) antibiotics, such as gentamycin or ampicillin; (7) cell protective agents, for example pluronic polyol; and (8) galactose. In one embodiment, soluble factors can be added to the culturing medium.

[0148] The mammalian cell culture that can be used with the present invention is prepared in a medium suitable for the type of cell being cultured. In one embodiment, the cell culture medium can be any one of those previously discussed (for example, MEM) that is supplemented with serum from a mammalian source (for example, fetal bovine serum (FBS)). In another embodiment, the medium can be a conditioned medium to sustain the growth of neuronal cells, brain cells, fibroblast cells, or hMN cells. In a further embodiment, neuronal cells, brain cells, fibroblast cells, or hMN cells, or any other mammalian cells, can be transfected with DNA vectors containing genes that encode a polypeptide or protein of interest. In other embodiments of the invention, cells are grown in a suspension culture (for example, a three-dimensional culture such as a hanging drop culture) in the presence of an effective amount of enzyme, wherein the enzyme substrate is an extracellular matrix molecule in the suspension culture. For example, the enzyme can be a hyaluronidase.

[0149] A suspension culture is a type of culture wherein cells, or aggregates of cells, multiply while suspended in liquid medium. A suspension culture comprising mammalian cells can be used for the maintenance of cell types that do not adhere or to enable cells to manifest specific cellular characteristics that are not seen in the adherent form. Some types of suspension cultures can include three-dimensional cultures or a hanging drop culture. A hanging-drop culture is a culture in which the material to be cultivated is inoculated into a drop of fluid attached to a flat surface (such as a coverglass, glass slide, Petri dish, flask, and the like), and can be inverted over a hollow surface. Cells in a hanging drop can aggregate toward the hanging center of a drop as a result of gravity. However, according to the methods of the invention, cells cultured in the presence of a protein that degrades the extracellular matrix (such as collagenase, chondroitinase, hyaluronidase, and the like) will become more compact and aggregated within the hanging drop culture, for degradation of the ECM will allow cells to become closer in proximity to one another since less of the ECM will be present. See also International PCT Publication No. W02007/100870, which is incorporated by reference.

[0150] Cells can be cultured as a single, homogenous population in a hanging drop culture, so as to generate an aggregate of cells, or can be cultured as a heterogeneous population in a hanging drop culture so as to generate a chimeric aggregate of cells.

[0151] Three-dimensional cultures can be formed from agar (such as Gey’s Agar), hydrogels (such as matrigel, agarose, and the like; Lee et al., (2004) Biomaterials 25: 2461-2466) or polymers that are cross-linked. These polymers can comprise natural polymers and their derivatives, synthetic polymers and their derivatives, or a combination thereof. Natural polymers can be anionic polymers, cationic polymers, amphipathic polymers, or neutral polymers. Non-limiting examples of anionic polymers can include hyaluronic acid, alginic acid (alginate), carageenan, chondroitin sulfate, dextran sulfate, and pectin. Some examples of cationic polymers include, but are not limited to, chitosan or polylysine. (Peppas et al., (2006 ) Adv Mater. 18: 1345-60; Hoffman, A. S., (2002) Adv Drug Deliv Rev . 43: 3-12; Hoffman, A. S., (2001) Ann NY Acad Sci 944: 62-73).

Examples of amphipathic polymers can include, but are not limited to collagen, gelatin, fibrin, and carboxymethyl chitin. Non-limiting examples of neutral polymers can include dextran, agarose, or pullulan. (Peppas et al., (2006 ) Adv Mater. 18: 1345-60; Hoffman, A. S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NY Acad Sci 944: 62-73).

[0152] Cells suitable for culturing according to methods of the invention can harbor introduced expression vectors, such as plasmids. The expression vector constructs can be introduced via transformation, microinjection, transfection, lipofection, electroporation, or infection. The expression vectors can contain coding sequences, or portions thereof, encoding the proteins for expression and production. Expression vectors containing sequences encoding the produced proteins and polypeptides, as well as the appropriate transcriptional and translational control elements, can be generated using methods well known to and practiced by those skilled in the art. These methods include synthetic techniques, in vitro recombinant DNA techniques, and in vivo genetic recombination which are described in J. Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and in F. M. Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.

[0153] Obtaining and Purifying Polypeptides

[0154] A polypeptide molecule encoded by a gene, or a variant thereof, can be obtained by purification from human cells expressing a protein or polypeptide encoded by a gene via in vitro or in vivo expression of a nucleic acid sequence encoding a protein or polypeptide; or by direct chemical synthesis.

[0155] Detecting Polypeptide Expression. Host cells which contain a nucleic acid encoding a protein or polypeptide, and which subsequently express a protein encoded by a gene, can be identified by various procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a nucleic acid encoding a protein or polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments of nucleic acids encoding the protein or polypeptide. In one

embodiment, a fragment of a nucleic acid of a gene can encompass any portion of at least about 8 consecutive nucleotides. In another embodiment, the fragment can comprise at least about 10 consecutive nucleotides, at least about 15 consecutive nucleotides, at least about 20 consecutive nucleotides, or at least about 30 consecutive nucleotides. Fragments can include all possible nucleotide lengths between about 8 and about 100 nucleotides, for example, lengths between about 15 and about 100 nucleotides, or between about 20 and about 100 nucleotides. Nucleic acid amplification-based assays involve the use of oligonucleotides to detect transformants which contain a nucleic acid encoding a protein or polypeptide.

[0156] Protocols for detecting and measuring the expression of a polypeptide encoded by a gene, using either polyclonal or monoclonal antibodies specific for the polypeptide are well established. Non-limiting examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal- based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a polypeptide encoded by a gene, can be used, or a competitive binding assay can be employed.

[0157] Labeling and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Methods for producing labeled hybridization or PCR probes for detecting sequences related to nucleic acid sequences encoding a protein, include, but are not limited to, oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.

[0158] Alternatively, nucleic acid sequences encoding a polypeptide encoded by a gene, can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, and/or magnetic particles.

[0159] Expression and Purification of Polypeptides. Host cells transformed with a nucleic acid sequence encoding a polypeptide, can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. Expression vectors containing a nucleic acid sequence encoding a polypeptide, can be designed to contain signal sequences which direct secretion of soluble polypeptide molecules encoded by a gene, or a variant thereof, through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound polypeptide molecules encoded by a gene or a variant thereof. [0160] Other constructions can also be used to join a gene sequence encoding a polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affmity purification system (Immunex Corp., Seattle, Wash.). Including cleavable linker sequences (i.e., those specific for Factor Xa or enterokinase (Invitrogen, San Diego, Calif.)) between the purification domain and a polypeptide encoded by a gene also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a polypeptide encoded by a gene and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by immobilized metal ion affinity chromatography, while the enterokinase cleavage site provides a means for purifying the polypeptide encoded by a gene.

[0161] A polypeptide can be purified from any human or non-human cell which expresses the polypeptide, including those which have been transfected with expression constructs that express a protein. A purified protein can be separated from other compounds which normally associate with a protein encoded by a gene in the cell, such as certain proteins, carbohydrates, or lipids, using methods practiced in the art. Non-limiting methods include size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.

[0162] Chemical Synthesis. Nucleic acid sequences comprising a gene that encodes a polypeptide can be synthesized, in whole or in part, using chemical methods known in the art.

Alternatively, a polypeptide, can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).

Optionally, fragments of polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule. In one embodiment, a fragment of a nucleic acid sequence that comprises a gene can encompass any portion of at least about 8 consecutive nucleotides. In one embodiment, the fragment can comprise at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, or at least about 30 nucleotides. Fragments include all possible nucleotide lengths between about 8 and about 100 nucleotides, for example, lengths between about 15 and about 100 nucleotides, or between about 20 and about 100

nucleotides. [0163] A fragment can be a fragment of a protein. For example, the fragment can encompass any portion of at least about 8 consecutive amino acids. The fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, a least about 50 consecutive amino acids, at least about 60 consecutive amino acids, at least about 70 consecutive amino acids, or at least about 75 consecutive amino acids. Fragments include all possible amino acid lengths between about 8 and 100 about amino acids, for example, lengths between about 10 and about 100 amino acids, between about 15 and about 100 amino acids, between about 20 and about 100 amino acids, between about 35 and about 100 amino acids, between about 40 and about 100 amino acids, between about 50 and about 100 amino acids, between about 70 and about 100 amino acids, between about 75 and about 100 amino acids, or between about 80 and about 100 amino acids.

[0164] A synthetic peptide can be substantially purified via high performance liquid

chromatography (HPLC). The composition of a synthetic polypeptide can be confirmed by amino acid analysis or sequencing. Additionally, any portion of an amino acid sequence comprising a protein encoded by a gene can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

[0165] Pharmaceutical Compositions and Administration for Therapy

[0166] Compounds of the invention can be administered to the subject once (e.g., as a single injection or deposition). Alternatively, compounds of the invention can be administered once or twice daily to a subject in need thereof for a period of from about two to about twenty-eight days, or from about seven to about ten days. Compounds of the invention can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof. Furthermore, compounds of the invention can be co-administrated with another therapeutic. Where a dosage regimen comprises multiple administrations, the effective amount of the compound(s) administered to the subject can comprise the total amount of the compound(s) administered over the entire dosage regimen.

[0167] Compounds can be administered to a subject by any means suitable for delivering the compounds to cells of the subject, such as brain tissue or neuronal cells. For example, compounds can be administered by methods suitable to transfect cells. Transfection methods for eukaryotic cells are well known in the art, and include direct injection of a nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor mediated nucleic acid delivery, bioballistic or particle acceleration; calcium phosphate precipitation, and transfection mediated by viral vectors. [0168] The compositions of this invention can be formulated and administered to reduce the symptoms associated with Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors by any means that produces contact of the active ingredient with the agent’s site of action in the body of a subject, such as a human or animal ( e.g ., a dog, cat, or horse). They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

[0169] The compounds of the invention may be administered to a subject in an amount effective to treat or prevent Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors. One of skill in the art can readily determine what will be an effective amount of the compounds of the invention to be administered to a subject, taking into account whether the compound is being used prophylactically or therapeutically, and taking into account other factors such as the age, weight and sex of the subject, any other drugs that the subject may be taking, any allergies or contraindications that the subject may have, and the like. For example, an effective amount can be determined by the skilled artisan using known procedures, including analysis of titration curves established in vitro or in vivo. Also, one of skill in the art can determine the effective dose from performing pilot experiments in suitable animal model species and scaling the doses up or down depending on the subjects weight etc. Effective amounts can also be determined by performing clinical trials in individuals of the same species as the subject, for example starting at a low dose and gradually increasing the dose and monitoring the effects on a neurodegenerative disorder. Appropriate dosing regimens can also be determined by one of skill in the art without undue experimentation, in order to determine, for example, whether to administer the agent in one single dose or in multiple doses, and in the case of multiple doses, to determine an effective interval between doses.

[0170] A therapeutically effective dose of a compound that treats or prevents Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors can depend upon a number of factors known to those of ordinary skill in the art. The dose(s) of the compounds can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the compound to have upon the target of interest. These amounts can be readily determined by a skilled artisan. These amounts include, for example, mg or microgram (pg) amounts per kilogram (kg) of subject weight, such as about 0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg or about 10 mg/kg, or between about 0.25 mg/kg to 0.5 mg/kg, 0.5 mg/kg to 1 mg/kg, 1 mg/kg to 2 mg/kg, 2 mg/kg to 3 mg/kg, 3 mg/kg to 4 mg/kg, 4 mg/kg to 5 mg/kg, 5 mg/kg to 6 mg/kg, 6 mg/kg to 7 mg/kg, 7 mg/kg to 8 mg/kg, 8 mg/kg to 9 mg/kg, or 9 mg/kg to 10 mg/kg, or any range in between. These amounts also include a unit dose of a compound, for example, mg or pg amounts, such as at least about 0.25 mg, 0.5 mg, 1 mg, 2 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg,

120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 225 mg, 250 mg,

275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg,

550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, or more. Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.

[0171] For compounds that are antagonists or inhibitors, or agonists, or activators, of a protein, or compounds that increase or decrease the expression of a gene or genes, the instructions would specify use of the pharmaceutical composition for treating or preventing a Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors.

[0172] Pharmaceutical compositions for use in accordance with the invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic compositions of the invention can be formulated for a variety of routes of

administration, including systemic and topical or localized administration. Techniques and formulations generally can be found in Remmington’s Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa (20^th Ed., 2000), the entire disclosure of which is herein incorporated by reference. For systemic administration, an injection is useful, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic compositions of the invention can be formulated in liquid solutions, for example in physiologically compatible buffers such as Hank’s solution or Ringer’s solution. In addition, the therapeutic compositions can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. These pharmaceutical formulations include formulations for human and veterinary use. [0173] According to the invention, a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any

conventional media or agent that is compatible with the active compound can be used.

Supplementary active compounds can also be incorporated into the compositions.

[0174] The invention also provides for a kit that comprises a pharmaceutically acceptable carrier and a compound identified using the screening assays of the invention packaged with instructions for use.

[0175] A pharmaceutical composition containing a compound of the invention can be administered in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed herein. Such pharmaceutical compositions can comprise, for example antibodies directed to polypeptides encoded by genes of interest or variants thereof, or agonists and antagonists of a polypeptide encoded by a gene of interest. The compositions can be administered alone or in combination with at least one other agent, such as a stabilizing compound, which can be

administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

[0176] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or

subcutaneous applications can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid;

buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0177] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions

(where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by

incorporating an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0178] Sterile injectable solutions can be prepared by incorporating the compound (e.g., a small molecule, peptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0179] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

[0180] Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0181] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.

Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In some embodiments, the compound can be applied via transdermal delivery systems, which slowly releases the active compound for percutaneous absorption. Permeation enhancers can be used to facilitate transdermal penetration of the active factors in the conditioned media. Transdermal patches are described in for example, U.S. Pat. No. 5,407,713; U.S. Pat. No. 5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat. No. 5,336,168; U.S. Pat. No. 5,290,561; U.S. Pat. No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S. Pat. No. 5,088,977; U.S. Pat. No. 5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No. 4,921,475.

[0182] Administration of the compound is not restricted to a single route, but may encompass administration by multiple routes. For instance, exemplary administrations by multiple routes include, among others, a combination of intradermal and intramuscular administration, or intradermal and subcutaneous administration. Multiple administrations may be sequential or concurrent. Other modes of application by multiple routes will be apparent to the skilled artisan.

[0183] The compounds of the invention may be formulated into compositions for administration to subjects for the treatment and/or prevention of Parkinson’s disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors. Such compositions may comprise the compounds of the invention in admixture with one or more pharmaceutically acceptable diluents and/or carriers and optionally one or more other pharmaceutically acceptable additives. The pharmaceutically- acceptable diluents and/or carriers and any other additives must be“acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the subject to whom the composition will be administered. One of skill in the art can readily formulate the compounds of the invention into compositions suitable for administration to subjects, such as human subjects, for example using the teaching a standard text such as Remington’s Pharmaceutical Sciences, 18th ed, (Mack Publishing Company: Easton, Pa., 1990), pp. 1635-36), and by taking into account the selected route of delivery.

[0184] Examples of diluents and/or carriers and/or other additives that may be used include, but are not limited to, water, glycols, oils, alcohols, aqueous solvents, organic solvents, DMSO, saline solutions, physiological buffer solutions, peptide carriers, starches, sugars, preservatives, antioxidants, coloring agents, pH buffering agents, granulating agents, lubricants, binders, disintegrating agents, emulsifiers, binders, excipients, extenders, glidants, solubilizers, stabilizers, surface active agents, suspending agents, tonicity agents, viscosity-altering agents, carboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate. The combination of diluents and/or carriers and/or other additives used can be varied taking into account the nature of the active agents used (for example the solubility and stability of the active agents), the route of delivery (e.g. oral, parenteral, etc.), whether the agents are to be delivered over an extended period (such as from a controlled-release capsule), whether the agents are to be co-administered with other agents, and various other factors. One of skill in the art will readily be able to formulate the compounds for the desired use without undue experimentation.

[0185] The compounds of the invention may be administered to a subject by any suitable method that allows the agent to exert its effect on the subject in vivo. For example, the

compositions may be administered to the subject by known procedures including, but not limited to, by oral administration, sublingual or buccal administration, parenteral administration, transdermal administration, via inhalation, via nasal delivery, vaginally, rectally, and intramuscularly. The compounds of the invention may be administered parenterally, or by epifascial, intracapsular, intracutaneous, subcutaneous, intradermal, intrathecal, intramuscular, intraperitoneal, intrasternal, intravascular, intravenous, parenchymatous, or sublingual delivery. Delivery may be by injection, infusion, catheter delivery, or some other means, such as by tablet or spray. In one embodiment, the compounds of the invention are administered to the subject by way of delivery directly to the brain tissue, such as by way of a catheter inserted into, or in the proximity of the subject’s brain, or by using delivery vehicles capable of targeting the drug to the brain. For example, the compounds of the invention may be conjugated to or administered in conjunction with an agent that is targeted to the brain, or the spinal cord, such as an antibody or antibody fragment. In one embodiment, the compounds of the invention are administered to the subject by way of delivery directly to the tissue of interest, such as by way of a catheter inserted into, or in the proximity of the subject’s tissue of interest, or by using delivery vehicles capable of targeting the drug to the brain, or the spinal cord, such as an antibody or antibody fragment.

[0186] For oral administration, a formulation of the compounds of the invention may be presented as capsules, tablets, powders, granules, or as a suspension or solution. The formulation may contain conventional additives, such as lactose, mannitol, cornstarch or potato starch, binders, crystalline cellulose, cellulose derivatives, acacia, cornstarch, gelatins, disintegrators, potato starch, sodium carboxymethylcellulose, dibasic calcium phosphate, anhydrous or sodium starch glycolate, lubricants, and/or or magnesium stearate.

[0187] For parenteral administration (z.e., administration by through a route other than the alimentary canal), the compounds of the invention may be combined with a sterile aqueous solution that is isotonic with the blood of the subject. Such a formulation may be prepared by dissolving the active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering the solution sterile. The formulation may be presented in unit or multi-dose containers, such as sealed ampoules or vials. The formulation may be delivered by injection, infusion, or other means known in the art.

[0188] For transdermal administration, the compounds of the invention may be combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, A-methyl pyrrol i done and the like, which increase the permeability of the skin to the compounds of the invention and permit the compounds to penetrate through the skin and into the bloodstream. The compounds of the invention also may be further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to provide the composition in gel form, which are dissolved in a solvent, such as methylene chloride, evaporated to the desired viscosity and then applied to backing material to provide a patch.

[0189] In some embodiments, the compounds of the invention are provided in unit dose form such as a tablet, capsule or single-dose injection or infusion vial.

[0190] Various routes of administration and various sites of cell implantation can be utilized, such as, subcutaneous, intramuscular, or in brain tissue, or neuronal tissue, in order to introduce aggregated population of cells into a site of preference. Once implanted in a subject (such as a mouse, rat, or human), the aggregated cells can then treat or prevent a neurodegenerative disorder within the subject. In one embodiment, transfected cells (for example, cells expressing a protein encoded by a gene) are implanted in a subject to treat or prevent Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors within the subject. In other embodiments, the transfected cells are cells derived from brain tissue. In further embodiments, the transfected cells are neuronal cells. Aggregated cells (for example, cells grown in a hanging drop culture) or transfected cells (for example, cells produced as described herein) maintained for 1 or more passages can be introduced (or implanted) into a subject (such as a rat, mouse, dog, cat, human, and the like). [0191] Subcutaneous” administration can refer to administration just beneath the skin (i.e., beneath the dermis). Generally, the subcutaneous tissue is a layer of fat and connective tissue that houses larger blood vessels and nerves. The size of this layer varies throughout the body and from person to person. The interface between the subcutaneous and muscle layers can be encompassed by subcutaneous administration.

[0192] Administration of the cell aggregates is not restricted to a single route, but can encompass administration by multiple routes. For instance, exemplary administrations by multiple routes include, among others, a combination of intradermal and intramuscular administration, or intradermal and subcutaneous administration. Multiple administrations can be sequential or concurrent. Other modes of application by multiple routes will be apparent to the skilled artisan.

[0193] In other embodiments, this implantation method will be a one-time treatment for some subjects. In further embodiments of the invention, multiple cell therapy implantations will be required. In some embodiments, the cells used for implantation will generally be subject-specific genetically engineered cells. In another embodiment, cells obtained from a different species or another individual of the same species can be used. Thus, using such cells can require

administering an immunosuppressant to prevent rejection of the implanted cells. Such methods have also been described in U.S. Patent Publication US 2004/0057937 and PCT Publication No. WO 2001/32840, and are hereby incorporated by reference.

[0194] Gene Therapy and Protein Replacement Methods

[0195] Delivery of nucleic acids into viable cells can be affected ex vivo, in situ, or in vivo by use of vectors, such as viral vectors (e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). Non-limiting techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, and the calcium phosphate precipitation method (See, for example, Anderson, Nature , supplement to vol. 392, no. 6679, pp. 25-20 (1998)). Introduction of a nucleic acid or a gene encoding a polypeptide of the invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells can also be cultured ex vivo in the presence of therapeutic compositions of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.

[0196] Nucleic acids can be inserted into vectors and used as gene therapy vectors. A number of viruses have been used as gene transfer vectors, including papovaviruses, e.g., SV40 (Madzak et al., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992; Quantin et al ., 1992; Rosenfeld et al, 1992; Wilkinson et al, 1992; Stratford-Perricaudet et al, 1990), vaccinia virus (Moss, 1992), adeno-associated virus (Muzyczka, 1992; Ohi et al ., 1990), herpesviruses including HSV and EBV (Margolskee, 1992; Johnson et al., 1992; Fink et al., 1992; Breakfield and Geller, 1987; Freese et al., 1990), and retroviruses of avian (Biandyopadhyay and Temin, 1984; Petropoulos et al., 1992), murine (Miller, 1992; Miller et al., 1985; Sorg et al, 1984; Mann and Baltimore, 1985; Miller et al., 1988), and human origin (Shimada et al., 1991; Helseth et al., 1990; Page et al., 1990; Buchschacher and Panganiban, 1992). Non-limiting examples of in vivo gene transfer techniques include transfection with viral ( e.g ., retroviral) vectors (see U.S.

Patent No. 5,252,479, which is incorporated by reference in its entirety) and viral coat protein- liposome mediated transfection (Dzau et al, Trends in Biotechnology 11 :205-2l0 (1993), incorporated entirely by reference). For example, naked DNA vaccines are generally known in the art; see Brower, Nature Biotechnology, 16: 1304-1305 (1998), which is incorporated by reference in its entirety. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g, Chen, et al, 1994. Proc. Natl. Acad. Sci. USA 91 : 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.

Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

[0197] For reviews of gene therapy protocols and methods see Anderson et al, Science

256:808-813 (1992); U.S. Patent Nos. 5,252,479, 5,747,469, 6,017,524, 6,143,290, 6,410,010 6,511,847; and U.S. Patent Publication Nos. US2002/0077313 and US2002/00069, which are all hereby incorporated by reference in their entireties. For additional reviews of gene therapy technology, see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); Miller, Nature, 357: 455-460 (1992); Kikuchi et al, J Dermatol Sci. 2008 May;50(2):87- 98; Isaka et al, Expert Opin Drug Deliv . 2007 Sep;4(5):56l-7l; Jager et al, Curr Gene Ther. 2007 Aug;7(4):272-83; Waehler et al, Nat Rev Genet. 2007 Aug;8(8):573-87; Jensen et al, AnnMed. 2007;39(2):l08-l5; Herweijer et al, Gene Ther. 2007 Jan;l4(2):99-l07; Eliyahu et al, Molecules, 2005 Jan 3 l;l0(l):34-64; and Altaras et al, Adv Biochem Eng Biotechnol . 2005;99: 193-260, all of which are hereby incorporated by reference in their entireties.

[0198] In some embodiments, the gene therapy is a CRISPR-based gene therapy. Introducing targeted modifications in the genome for therapeutic purposes, can require highly efficient systems that are able to alter the existing DNA pattern with great precision. The CRISPR/Cas9 type II system consists of the Cas9 nuclease and a single guide RNA (sgRNA or gRNA), which is a fusion of a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) that binds Cas9 nuclease and directs it to a target sequence based on a complementary base-pairing rule. The target sequence must be adjacent to a protospacer-adjacent motif (PAM) consisting of a canonical NGG or NAG sequence. At the recognition site, a double-strand break (DSB) is generated that can be repaired by non-homologous end joining (NHEJ), resulting in small insertions or deletions usually associated with loss of function (knockdown/knockout). In the presence of an exogenous donor DNA, by a homology directed recombination (HDR) mechanism, precise modifications can be achieved at the targeted site, resulting in gain of function (knockin). For additional review of CRISPR technology, see Chira el al, Mol. Thr. Nuc. Acids. 2017, Vol. 7, p2l 1, the contents of which is hereby incorporated by reference in its entirety.

[0199] Protein replacement therapy can increase the amount of protein by exogenously introducing wild-type or biologically functional protein by way of infusion. A replacement polypeptide can be synthesized according to known chemical techniques or can be produced and purified via known molecular biological techniques. Protein replacement therapy has been developed for various disorders. For example, a wild-type protein can be purified from a recombinant cellular expression system ( e.g ., mammalian cells or insect cells -see U.S. Patent No. 5,580,757 to Desnick et al:, U.S. Patent Nos. 6,395,884 and 6,458,574 to Selden et al:, U.S. Patent No. 6,461,609 to Calhoun et al .; U.S. Patent No. 6,210,666 to Miyamura et al:, U.S. Patent No. 6,083,725 to Selden et al .; U.S. Patent No. 6,451,600 to Rasmussen et al .; U.S. Patent No.

5,236,838 to Rasmussen et al. and U.S. Patent No. 5,879,680 to Ginns et al .), human placenta, or animal milk (see U.S. Patent No. 6,188,045 to Reuser et al .), or other sources known in the art.

After the infusion, the exogenous protein can be taken up by tissues through non-specific or receptor-mediated mechanism.

[0200] A polypeptide encoded by a gene of interest, for example, but not limited to, SNX25 and

SNX27 can also be delivered in a controlled release system. For example, the polypeptide can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump can be used (see, i.e.,

Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery

88:507 (1980); Saudek et al., N. Engl. J. Med. 321 :574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.),

CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and

Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol.

Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228: 190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71 : 105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).

[0201] Combination Therapy

[0202] According to the methods of the invention, a compound of the invention can be administered to a subject either as a single agent, or in combination with one or more other agents. In one embodiment, a compound of the invention is administered to a subject as a single agent. In one embodiment, a compound of the invention is administered to a subject alone. In one embodiment, a compound of the invention is administered to a subject in combination with one or more other agents.

[0203] In certain embodiments, a compound of the invention may be used in combination with other agents that are used for the treatment or prevention of Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors. In certain embodiments, a compound of the invention may be used in combination with other agents that are not used for the treatment or prevention of Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors. In one embodiment, a compound of the invention may be delivered to a subject as part of the same pharmaceutical composition or formulation containing one or more additional active agents. In another embodiment, a compound of the invention may be delivered to a subject in a composition or formulation containing only that active agent, while one or more other agents are administered to the subject in one or more separate compositions or formulations. In one embodiment, the other agents are not used for the treatment or prevention of Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors. In another embodiment, the other agents are used for the treatment or prevention of Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors.

[0204] A compound of the invention and the other agents that are used for the treatment or prevention of Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors may be administered to the subject at the same time, or at different times. A compound of the invention and the other agents that are not used for the treatment or prevention of Parkinson’s

Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors may be administered to the subject at the same time, or at different times. For example, a compound of the invention and the other agents may be administered within minutes, hours, days, weeks, or months of each other, for example as part of the overall treatment regimen of a subject. In some embodiments, a compound of the invention may be administered prior to the administration of other agents. In other embodiments, a compound of the invention may be administered subsequent to the administration of other agents.

[0205] A compound of the invention may also be used in combination with known therapies for

Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors. A compound of the invention may also be used in combination with surgical or other interventional treatment regimens used for the treatment of Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors.

[0206] Compounds of the invention, as described above, including, but not limited to, inhibitors, activators, agonists, and antagonists, may be used in combination with each other for the treatment or prevention of Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors.

[0207] In some embodiments, the administration of a compound of the invention in

combination with one or more other agents has an additive effect, in comparison with

administration of the compound of the invention alone, or administration of the one or more other agents alone. In other embodiments, the administration of a compound of the invention in combination with one or more other agents has a synergistic effect, in comparison with

administration of the compound of the invention alone, or administration of the one or more other agents alone. In some embodiments, the administration of a compound of the invention in combination with one or more other agents can help reduce side effects, in comparison with administration of the compound of the invention alone, or administration of the one or more other agents alone.

[0208] In some embodiments, the compound of the invention is used as an adjuvant therapy. In other embodiments, the compound of the invention is used in combination with an adjuvant therapy.

[0209] Subjects

[0210] According to the methods of the invention, the subject or patient can be any animal that has or is diagnosed with Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors. According to the methods of the invention, the subject or patient can be any animal that is predisposed to or is at risk of developing Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors. In preferred embodiments, the subject is a human subject. In some embodiments, the subject is a rodent, such as a mouse. In some embodiments, the subject is a cow, pig, sheep, goat, cat, horse, dog, and/or any other species of animal used as livestock or kept as pets. [0211] In some embodiments, the subject is already suspected to have Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors. In other embodiments, the subject is being treated for Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors, before being treated according to the methods of the invention. In other embodiments, the subject is not being treated for Parkinson’s Disease and/or lysosomal toxicity caused by LRRK2 kinase inhibitors, before being treated according to the methods of the invention.

[0212] In some embodiments, the subject has one or more mutations in LRRK2. In some embodiments, the one or more mutations in LRRK2 is G2019S, I2020T, R1441C/G/H, or Y1699C. In some embodiment, the subject is homozygous for an allele of LRRK2 associated with

Parkinson’s Disease. In some embodiment, the subject is heterozygous for an allele of LRRK2 associated with Parkinson’s Disease. In some embodiments, the subject has one or more single nucleotide polymorphisms (SNP) in LRRK2 associated with Parkinson’s Disease.

EXAMPLES

[0213] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

[0214] Example 1: hMN cells assays and tools

[0215] Human IPSC-derived motor neurons were used in these studies, and perform the analysis of lysosomes using a completely automated 96-well high-content imaging system.

Lysosome puncta size is quantified in terms of either the uptake of fluorescent dextran, or by lysotracker staining, within cells (identified using Cellmask and a nuclear staining dye).

[0216] The cell type used was human IPSC-derived motor neurons. For imaging the following was used: Cell Mask: Plasma membrane Stain (Deep Red); Dextran: (endocytic trafficking) Alexa 488 (Green); Lysotracker: (lysosome morphology and polarization)Red DND-99 (Red).

[0217] Fully automated imaging and analysis was performed in a 384-well format. Analysis tools identify cell outline and quantify size of dextran- or lysotracker-positive puncta.

[0218] IN Cell Analyzer 2000: The data was gathered using a high-throughput imaging system for performing high-content analysis. An IN Cell Analyzer 2000 was used (GE

Healthcare) equipped with a large CCD camera (resolution 2048 x 2048 pixels) that is capable of whole-well imaging in 96-well microplates. [0219] High-content image analysis was performed by the IN Cell Investigator software. It includes several image analysis modules (Object Intensity, Nuclear Trafficking, Plasma Membrane Trafficking, Granularity, Cell Cycle Trafficking, Morphology, Dual Object, Neurite Outgrowth, Micro Nuclei, Multi-Target Analysis).

[0220] Using the IN-Cell Investigator software, Cells defined by DAPI (nuclei) and cell mask (Cy5). Organelles were defined for each cell on both channels (FITC and DsRed). Organelle 1 - FITC (dextran), Organelle 2 - DsRED (LysoTracker).

[0221] FIG. 1 shows the protocol used in the hMN cell assay. FIG. 2 shows imaging of cell mask, lysotracker, and dextran in hMN cells treated with lOnm or lOOnm CZC-25146.

[0222] CRISPR-SAM technology was used to induce expression of LRRK2 or various SNX proteins, as validated by qRNA for efficacy and selectivity (FIG. 3). For some targets such as LRRK2, protein induction was validated by Western blot. Background intensity and cell numbers are thus normalized across fields in an automated fashion. FIG. 4 shows the efficiency of SAM tools (RT-qPCR) in SY5Y. N=3 per well. Overexpression was specific for the target of each guide.

[0223] The dose dependent effect of the LRRK2 inhibitor CZC-25146 on lysosome puncta size using either lysotracker or dextran uptake as readouts was established (FIGS. 5-8). At the lower dose used (lOnM), there was a trend of an effect of the inhibitor alone on lysosome puncta size that did not reach significance by dextran analysis, whereas this effect was significant at the higher dose (lOOnM). FIG. 5 shows LRRK2 kinase activity may suppress other effector functions of LRRK2. Induction of LRRK2 expression alone (~5 fold) did not significantly effect lysosome puncta size as quantified by lysotracker or dextran, in the absence of LRRK2 inhibitor. But remarkably, induction of LRRK2 expression led to highly significant effect on lysosome puncta size in the presence of the LRRK2 inhibitor, even at the low inhibitor dose. Thus, the effect of the LRRK2 inhibitor on puncta size is synergistic with the effect of the induction of endogenous LRRK2 expression. One interpretation of these findings is that the LRRK2 kinase domain actually suppresses other LRRK2 activities within this multicomponent protein, and that these other activities drive the increased lysosome size in the presence of the kinase inhibitor. FIG. 6 shows similar results with Lysotracker analysis.

[0224] FIG. 7 shows that at higher dose (lOOnM, in green), CZC LRRK2 inhibitor alone is sufficient to increase lysosome size as quantified here by dextran imaging, consistent with other model systems reported. The effect of increasing LRRK2 expression by CRISPR-SAM appears additive or synergistic with the effect of the inhibitor, again suggesting that the effect of LRRK2 inhibitor is in part through the disinhibition of a LRRK2 activity other than the kinase activity. The effect of increasing LRRK2 expression by CRISPR-SAM appears synergistic with inhibitor at the lower dose, but may plateau at highest dose (lOOnM, in green). FIG. 8 shows similar results with Lysotracker analysis.

[0225] Induction of SNX3 leads to increased lysosome puncta size, and this effect appears synergistic with the LRRK2 inhibitor effect on increasing puncta size (SNX3 induction thus essentially phenocopies the effect of LRRK2 induction), suggesting that these genes function in a common pathway. FIG. 9 shows analysis of the effect of SNX gene induction on lysosomes in hMNs. By lysotracker analysis: SNX3 induction essentially phenocopies LRRK2 induction, as the effect is mostly seen in the context of LRRK2 inhibitor treatment. SNX25 or SNX27 induction appears to suppress the effect of the LRRK2 kinase inhibitor on lysosome size. These data support the idea that SNX proteins function in a common pathway with LRRK2.

[0226] FIG. 10 shows analysis of effect of SNX gene induction on lysosomes in hMNs by dextran analysis. SNX3 induction leads overall to increased lysosome puncta size. SNX25 induction appears to suppress the effect of the LRRK2 kinase inhibitor on lysosome size (whereas the effect of SNX27 induction shows a similar trend but does not reach statistical significance). These data support the idea that SNX proteins function in a common pathway with LRRK2.

Induction of SNX25 or SNX27 expression appear to selectively suppress the effect of the LRRK2 kinase inhibitor on lysosome puncta size.

[0227] These studies suggest that the well -reported lysosomal toxicity of LRRK2 kinase inhibitors, in vivo and in vitro, may be due to disinhibition of other LRRK2 activities within this multicomponent protein and underscore the need for a further understanding of the role of other domains. The data support a differential role for different SNX proteins, and support a relationship between the functions of SNX proteins and of LRRK2. It is possible that either LRRK2 deficiency, or excess LRRK2 in the presence of an inhibitor, lead to enlarged lysosome puncta. Further repeat studies with other LRRK2 inhibitors can be performed. The domains or activities of LRRK2 that are relevant here can be tested. Combination induction of LRRK2 and SNX proteins can be studied to pursue epistasis. The identification of other genes/proteins and drugs that suppress LRRK2 kinase inhibitor phenotype can be performed.

Claims

What is claimed is:

1. A method of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a nucleic acid encoding SNX25, a nucleic acid encoding SNX27, or a combination thereof.

2. A method of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a SNX25 polypeptide, a SNX27 polypeptide, or a combination thereof.

3. A method of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a SNX25 activator or agonist, a SNX27 activator or agonist, or a combination thereof.

4. The method of claims 1, 2, or 3, wherein the lysosomal toxicity is enlarged lysosomal

puncta.

5. The method of claims 1, 2, or 3, wherein the LRRK2 kinase inhibitor is LRRK2-IN-1, CZC- 54252, CZC-25146, HG-10-102-1, GSK2578215A, JH-II-127, GNE-7915, GNE-0877, GNE-9605, PF-06447475, or MLi-2.

6. The method of claims 1, 2, or 3, wherein the subject has one or more mutations in LRRK2.

7. The method of claim 6, wherein the one or more mutations in LRRK2 is G2019S, I2020T, R1441C/G/H, or Y1699C.

8. The method of claims 1, 2, or 3, wherein the subject has one or more single nucleotide

polymorphisms (SNP) in LRRK2 associated with Parkinson’s Disease.

9. The method of claims 1, 2, or 3, wherein the subject has Parkinson’s Disease.

10. The method of claims 1, 2, or 3, wherein the subject is at risk of developing Parkinson’s Disease.

11. A method of treating Parkinson’s Disease in a subject in need thereof comprising

administering to the subject a therapeutically effective amount of LRRK2 kinase inhibitor and a therapeutically effective amount of a nucleic acid encoding SNX25, a nucleic acid encoding SNX27, or a combination thereof.

12. A method of treating Parkinson’s Disease in a subject in need thereof comprising

administering to the subject a therapeutically effective amount of LRRK2 kinase inhibitor and a therapeutically effective amount of a SNX25 polypeptide, a SNX27 polypeptide, or a combination thereof.

13. A method of treating Parkinson’s Disease in a subject in need thereof comprising

administering to the subject a therapeutically effective amount of LRRK2 kinase inhibitor and a therapeutically effective amount of a SNX25 activator or agonist, a SNX27 activator or agonist, or a combination thereof.

14. The method of claims 11, 12, or 13, wherein the LRRK2 kinase inhibitor causes lysosomal toxicity.

15. The method of claim 14, wherein the lysosomal toxicity is enlarged lysosomal puncta.

16. The method of claims 11, 12, or 13, wherein the LRRK2 kinase inhibitor is LRRK2-IN-1, CZC-54252, CZC-25146, HG-10-102-1, GSK2578215A, JH-II-127, GNE-7915, GNE- 0877, GNE-9605, PF-06447475, or MLi-2.

17. The method of claims 11, 12, or 13, wherein the subject has one or more mutations in

LRRK2.

18. The method of claim 17, wherein the one or more mutations in LRRK2 is G2019S, I2020T, R1441C/G/H, or Y1699C.

19. The method of claims 11, 12, or 13, wherein the subject has one or more single nucleotide polymorphisms (SNP) in LRRK2 associated with Parkinson’s Disease.

20. A method of screening for a compound that inhibits, treats, or prevents LRRK2 kinase inhibitor lysosomal toxicity, the method comprising: a. providing a cell line in cell culture; b. contacting the cells with a LRRK2 inhibitor; c. contacting the cells with a compound; d. contacting the cells with fluorescently labeled dextran; e. contacting the cells with a fluorescent plasma membrane stain; f. imaging the cells, wherein the fluorescently labeled dextran and/or the fluorescently labeled plasma membrane are imaged; g. quantifying the average size of dextran vesicles in the cells; and h. comparing the average size of dextran vesicles in the cells to the average size of dextran vesicles in cells of a control cell culture that was not contacted with the compound, wherein a reduction in the average size of dextran vesicles in the cells is indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

21. A method of screening for a compound that inhibits, treats, or prevents LRRK2 kinase inhibitor lysosomal toxicity, the method comprising: a. providing a cell line in cell culture; b. contacting the cells with a LRRK2 inhibitor; c. contacting the cells with a compound; d. contacting the cells with a fluorescent dye for labeling lysosomes; e. contacting the cells with a fluorescent plasma membrane stain; f. imaging the cells, wherein the fluorescently labeled lysosomes and/or the

fluorescently labeled plasma membrane are imaged; g. quantifying the average size of lysosomal vesicles in the cells; and h. comparing the average size of lysosomal vesicles in the cells to the average size of lysosomal vesicles in cells of a control cell culture that was not contacted with the compound, wherein a reduction in the average size of lysosomal vesicles in the cells is indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

22. The method of claim 20 or 21, wherein the cell line has an increased level of LRRK2.

23. The method of claim 22, wherein CRISPR is used to increase the level of LRRK2 in the cell line.

24. The method of claim 20 or 21, wherein the cell line in cell culture is human motor neuron cells.

25. The method of claim 20 or 21, wherein the cell line is cultured in a multiwell cell culture plate and wherein one or more compounds are screened, wherein each compound is applied to a separate well of the multiwell cell culture plate.

26. The method of claim 20, further comprising contacting the cells with a fluorescent dye for labeling lysosomes in step d).

27. The method of claim 26, further comprising imaging the fluorescently labeled lysosomes in step f), quantifying the average size of lysosomal vesicles in the cells; and comparing the average size of lysosomal vesicles in the cells to the average size of lysosomal vesicles in cells of a control cell culture that was not contacted with the compound, wherein a reduction in the average size of lysosomal vesicles in the cells is further indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

28. The method of claim 21, further comprising contacting the cells with a fluorescently labeled dextran in step d).

29. The method of claim 28, further comprising imaging the fluorescently labeled dextran in step f), quantifying the average size of dextran vesicles in the cells; and comparing the average size of dextran vesicles in the cells to the average size of dextran vesicles in cells of a control cell culture that was not contacted with the compound, wherein a reduction in the average size of dextran vesicles in the cells is further indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

30. The method of claims 20 or 21, wherein the LRRK2 kinase inhibitor is LRRK2-IN-1, CZC- 54252, CZC-25146, HG-10-102-1, GSK2578215A, JH-II-127, GNE-7915, GNE-0877, GNE-9605, PF-06447475, or MLi-2.

31. A method of screening for a gene or protein that inhibits, treats, or prevents LRRK2 kinase inhibitor lysosomal toxicity, the method comprising: a. providing a cell line in cell culture; b. increasing or decreasing the level of a gene or protein in the cell line; c. contacting the cells with a LRRK2 inhibitor; d. contacting the cells with fluorescently labeled dextran; e. contacting the cells with a fluorescent plasma membrane stain; f. imaging the cells, wherein the fluorescently labeled dextran and/or the fluorescently labeled plasma membrane are imaged; g. quantifying the average size of dextran vesicles in the cells; and h. comparing the average size of dextran vesicles in the cells to the average size of dextran vesicles in cells of a control cell culture wherein the level of the gene or protein was not increased or decreased, wherein a reduction in the average size of dextran vesicles in the cells is indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

32. A method of screening for a gene or protein that inhibits, treats, or prevents LRRK2 kinase inhibitor lysosomal toxicity, the method comprising: a. providing a cell line in cell culture; b. increasing or decreasing the level of a gene or protein in the cell line; c. contacting the cells with a LRRK2 inhibitor; d. contacting the cells with a fluorescent dye for labeling lysosomes; e. contacting the cells with a fluorescent plasma membrane stain; f. imaging the cells, wherein the fluorescently labeled lysosomes and/or the

fluorescently labeled plasma membrane are imaged; g. quantifying the average size of lysosomal vesicles in the cells; and h. comparing the average size of lysosomal vesicles in the cells to the average size of lysosomal vesicles in cells of a control cell culture wherein the level of the gene or protein was not increased or decreased, wherein a reduction in the average size of lysosomal vesicles in the cells is indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

33. The method of claim 31 or 32, wherein the cell line has an increased level of LRRK2.

34. The method of claim 33, wherein CRISPR is used to increase the level of LRRK2 in the cell line.

35. The method of claim 31 or 32, wherein the cell line in cell culture is human motor neuron cells.

36. The method of claim 31 or 32, wherein the cell line is cultured in a multiwell cell culture plate and wherein one or more compounds are screened, wherein each compound is applied to a separate well of the multiwell cell culture plate.

37. The method of claim 31, further comprising contacting the cells with a fluorescent dye for labeling lysosomes in step d).

38. The method of claim 37, further comprising imaging the fluorescently labeled lysosomes in step f), quantifying the average size of lysosomal vesicles in the cells; and comparing the average size of lysosomal vesicles in the cells to the average size of lysosomal vesicles in cells of a control cell culture wherein the level of the gene or protein was not increased or decreased, wherein a reduction in the average size of lysosomal vesicles in the cells is further indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

39. The method of claim 32, further comprising contacting the cells with a fluorescently labeled dextran in step d).

40. The method of claim 39, further comprising imaging the fluorescently labeled dextran in step f), quantifying the average size of dextran vesicles in the cells; and comparing the average size of dextran vesicles in the cells to the average size of dextran vesicles in cells of a control cell culture wherein the level of the gene or protein was not increased or decreased, wherein a reduction in the average size of dextran vesicles in the cells is further indicative of inhibiting, treating, or preventing LRRK2 kinase inhibitor lysosomal toxicity.

41. The method of claim 31 or 32, wherein CRISPR is used to increase or decrease the level of the gene or protein in the cell line.

42. The method of claims 31 or 32, wherein the LRRK2 kinase inhibitor is LRRK2-IN-1, CZC- 54252, CZC-25146, HG-10-102-1, GSK2578215A, JH-II-127, GNE-7915, GNE-0877, GNE-9605, PF-06447475, or MLi-2.